MANUAL

54

TABLE OF CONTENTS

1.0 INFLAMMATION

1.1 Purification of mononuclear and polymorphonuclear cells4

1.2 Isolation of cells from the peritoneal cavities of mice8

1.3 Adherence purification of monocytes9

1.4 Antibody- or C3b-dependent phagocytosis10

1.5 Activation of macrophages12

1.6 Monokine Bioassays14

1.6.1 Assay for IL-1 activity14

1.6.2 Assay for IL-6 activity17

1.6.3 Cytotoxicity assay for TNFa19

1.7. Neutrophil chemotaxis assay21

1.8. FceRI-dependent activation of mast cells24

2.0 ANTIBODIES: PURIFICATION & CHARACTERIZATION

2.1 Hybridoma cell culture with production of monoclonal antibodies26

2.2 Affinity purification of IgG antibodies27

2.2.1 Avid-AL affinity chromatography27

2.2.2 Protein A-Sepharose affinity chromatography29

2.3 Preparation of IgM antibodies31

2.4 Analysis & characterization of immunoglobulins33

2.4.1 Polyacrylamide gel electrophoresis of immunoglobulins33

2.4.2 Western blotting to detect immunoglobulins35

3.0 T CELL AND B CELL RESPONSES

3.1 C'-dependent depletion of CD4+ and CD8+ T Cells37

3.2 MACS purification (or depletion) of CD4+ and CD8+ T Cells39

3.3 Assessment of T cell proliferation41

3.4 Plaque Forming Cell (PFC) assay for IgM-producing cells44

3.5 ELISPOT assays for single cytokine- or Ab-producing cells45

3.6 ELISA assay for detection of antigen-specific antibodies49

3.7 In vivo assessment of T cell responses51

3.8 Immunohistochemical detection of cytokines in tissues53

4.0 MOLECULAR ANALYSIS OF CYTOKINE mRNA EXPRESSION

4.1 Northern blotting55

4.1.1 Purification of cellular RNA55

4.1.2 Electrophoresis of RNA & transfer to membranes58

4.1.3 32P-labelled cDNA probe synthesis61

4.1.4 Pre-hybridization, hybridization, and washing63

4.1.5 Detection of mRNA bands65

4.2 In Situ hybridization67

4.2.1 Probe synthesis & purification67

4.2.2 Preparation of slides for hybridization71

4.2.3 Hybridization of 35S-cRNA riboprobes to cellular mRNA74

4.2.4 Post-hybridization washing & autoradiography76

4.2.5 Autoradiograph development & counter-staining78

4.3 Semi-quantitative RT-PCR to detect cytokine mRNA79

4.3.1 First strand cDNA Synthesis using Oligo(dT) priming80

4.3.2 PCR amplification of the target cDNA81

4.3.2 Detection of RT-PCR products82

APPENDICES

5.1 APPENDIX A -- GENERAL METHODS83

5.1.1 Anti-sheep RBC antisera83

5.1.2 Cell counting83

5.1.3 C3b opsinization of yeast84

5.1.4 Cytocentrifuge preparations85

5.1.5 Dialysis tubing85

5.1.6 Fixation of tissues for ISH or IHC85

5.1.7 Lung cells (single cell suspension)86

5.1.8 Lysis of red blood cells86

5.1.8.1 Hypotonic lysis with H2O86

5.1.8.2 Lysis with ammonium chloride86

5.1.9 Opsinization of SRBC with antibody87

5.4.10 Protein assay in microtiter plates87

5.1.11 Splenocytes (single cell suspensions)87

5.1.12 Splenocytes (spleen cell-conditioned medium)88

5.1.13 Staining Protocols89

5.1.13.1 Giemsa stains89

5.1.13.1.1 Wrights-Giemsa staining89

5.1.13.1.2 Giemsa staining of tissue sections89

5.1.13.2 Gills hematoxylin for IHC90

5.1.13.3 Toluidine blue staining (ISH counter-stain)90

5.1.14 Standard curves (e.g., cytokines)90

5.1.15 TESPA-treatment of glass slides91

5.2 APPENDIX B -- REAGENTS & SOLUTIONS

CELLULAR IMMUNOLOGY REAGENTS92

Acidified isopropanol92

Actinomycin D92

Alsevers solution92

Ammonium chloride92

Ammonium sulfate (saturated solutions)92

Borate-buffered saline92

ELISPOT & ELISA Carbonate Coating buffer92

Isotonic Percoll Density Gradient Medium93

PAGE running buffer93

PAGE 2x sample prep buffer93

PAGE gel fix buffer93

Phosphate-buffered saline (PBS)93

Giemsa Stain93

Giemsa Stock Solution93

0.4% Trypan Blue94

MOLECULAR BIOLOGY REAGENTS

Agarose/formaldehyde/MOPS gel (for Northerns)94

Cesium Chloride (for isolation of total cellular RNA)94

DEPC-treated water (& other solutions)94

Dithiothreitol94

EDTA (0.5M)94

Guanidinium Isothiocyanate (GSCN)94

ISH 10x salts94

ISH hybridization buffer95

Northern blotting pre-hyb/hybridization solution95

MOPS (1 M)95

5X MOPS Buffer95

Phenol (salt-saturated)95

Reagents for purifying DNA from agarose gels96

General purpose restriction endonuclease buffers96

RNA sample prep buffer (Northern analysis)96

RNA sample dye/loading buffer96

RNAse A96

Salmon sperm DNA96

Sodium acetate (3M)96

20X SSC (4 liters)97

STE buffer97

5.3 APPENDIX C -- TISSUE CULTURE MEDIA

Click's medium98

DMEM98

DMEM-0% FCS98

DMEM-10% FCS.98

DMEM-10% normal horse serum98

HBSS (Ca++ and Mg++-free)98

MEM98

RPMI 164098

RPMI-0% FCS.98

RPMI-10% FCS98

5.4 APPENDIX D -- MAINTENANCE OF CELL LINES

7TD1 cells (for assay of IL-6)99

Cl.MC/C57.1 cells (C57 mast cells)99

L-929 cells (for TNF bioassay)99

LM-1 cells (for assay of IL-1)99

Pu5-1.8 cells (macrophage cell line)99

5.6 APPENDIX F -- Human cytokine RT-PCR primers101

5.7 APPENDIX G -- WWW Immunology sites of interest102

5.7 APPENDIX G -- Selected templates for 96--well plates103

1.0 INFLAMMATION: TC "1.0 INFLAMMATION:" \l 1 \n

In the first week of this course we will begin to acquire some of the basic skills needed to examine several components of the inflammatory cascade, a series of responses that are integrally intertwined with the immune system. First we will begin to acquire some basic skills in tissue culture, and the purification of selected cells associated with the immunoinflammatory system (i.e., peripheral blood [PBL] monocytes and neutrophils XE "neutrophils " [PMN XE "PMN " or polymorphonuclear cells XE "polymorphonuclear cells" ]). Then we will learn how to identify them using morphologic criteria, and how to assess a couple of functions associated with monocytes/macrophages - phagocytosis (a subfunction of antibody-dependent cellular cytotoxicity) and activation-dependent monokine production.

1.1 Purification of mononuclear and polymorphonuclear cells TC "1.1 Purification of mononuclear and polymorphonuclear cells" \l 2 from mouse peripheral blood.

While this protocol is designed specifically for the purification of cells from the peripheral blood XE "peripheral blood" , such density gradient systems XE "density gradient systems" can also be used for other cell systems (e.g., to purify PMN from glycogen-elicited peritoneal cavity preparations). In general it is better to use polypropylene [p.p.] rather than polystyrene [p.s.] tubes because polypropylene is less "sticky" for the cells and therefore activates them to a lesser extent than polystryrene. On the other hand, polypropylene tubes are more expensive, so this too needs to be taken into account.

Materials

BALB/c mouse

anaesthetic XE "anaesthetic" (methoxyfluorane)

clinical centrifuge, 15 ml centrifuge tubes, 4 ml round bottom p.s. tubes

23-25 ga needles & 1 ml syringes

pipettes/pipettors (micro- and macropipettes)

microscope slides (frosted end; & pencil for labeling)

sterile surgical tools (optional, for open body cardiac puncture)

laminar flow hood, inverted microscope, hemocytometer

Reagents

anticoagulant XE "anticoagulant" (heparin XE "heparin" 1000U/ml)DMEM with 1 U/ml heparin or with 3 mg/ml EDTA anticoagulantDMEM-10% FCS (see Appendix A)

70% ethanol

isotonic Percoll XE "Percoll" (see Appendix C)

RBC sedimentation buffer XE "RBC sedimentation buffer" (4.5% dextran-T500 in PBS)

cytocentrifuge

METHOD

1.Obtain blood from surgically anaesthetized (or euthanized) mouse either by closed- or open-body cardiac puncture. Withdraw blood slowly into a 1 ml syringe containing 100 l of heparin, being careful not to hemolyse the red blood cells by using too much back pressure on syringe. Work fairly quickly, or mix the blood with the anticoagulant in the syringe fairly often, so that the blood doesn't coagulate.

2.Mix the blood with the RBC sedimentation buffer (1 volume sed. buffer: 1 volume blood) in the 4 ml tubes and allow the mixture to stand undisturbed on the bench for 20 - 45' (the time can be highly variable, depending on the species of animal). The RBC's will form rouleaux XE "rouleaux " (chains of RBC's) that will sediment out of suspension rather rapidly, leaving you with a leukocyte/platelet-rich plasma XE "leukocyte/platelet-rich plasma" layer above the settled RBC's.

3.Withdraw the plasma layer and transfer to a 15 ml polypropylene tube. Add 10 - 12 ml of DMEM-anticoagulant to the plasma and sediment the leukocytes out of suspension by centrifugation (1000 rpm for 10'),. Aspirate the platelet XE "platelet" -rich medium, avoiding the cell pellet, and then resuspend the cells by vigorously flicking the tube. When the cell pellet has become a 'paste' on the lower walls of the tube, add 5 - 10 ml of tissue culture medium and then gently vortex by hand to ensure that you have a 'single cell suspension' (i.e., no clumps of cells).

4.While the cells are spinning in step 3, prepare a 70% isotonic Percoll XE "isotonic Percoll" gradient ;cushion by mixing together 1.75 ml isotonic Percoll and 0.75 ml of DMEM/heparin/EDTA, in a 15 ml p.p. tube. After resuspending the cell pellet from step 3, gently layer this suspension on top of the 70% isotonic Percoll 'cushion', so that there is no mixing of the two layers. Centrifuge the cell gradients for 25 min at 2000 rpm (room temperature) in the clinical centrifuge, and allow the centrifuge rotor to coast to a stop (i.e., no brake).

5.The completed gradient will appear much as it did before spinning, but now the PMN and RBC will reside as a pellet at the bottom of the tube and the mononuclear cells (monocytes and lymphocytes) will reside as a band of cells at the interface between the tissue culture medium and the isotonic Percoll. To harvest the mononuclear cells, simply pipette the cells directly from the interface, trying to avoid aspirating any substantial volume of the Percoll. Transfer the cells to a new tube and dilute any of the density gradient medium by topping up the tube with additional medium. To harvest the PMN, carefully aspirate the remaining Percoll solution, then resuspend the pelleted cells by flicking the tube and then adding a large volume of medium. If the PMN are badly contaminated with red blood cells, these can by lysed by hypotonic lysis or ammonium chloride (see Appendix D).

6.Wash both cell preparations (i.e., PMN and mononuclear cells) as above, and resuspend the cells in a minimal volume (e.g., 1 ml) of medium. Remove 20 l of each preparation for cell counting by hemocytometer and determine the cell numbers and viabilities (see Appendix D). If the mononuclear cell fraction is heavily contaminated with platelets, rewash at low speed (i.e., 800 rpm for 10').

7.Prepare cytocentrifuge slides of the cells by applying 5x104 cells in 100 l of medium to each slide assembly and centrifuging them for 5 min at 1500 rpm. Allow the sedimented cells to dry well before staining them with Giemsa solution (see Appendix D).

8.If you have sufficient cells remaining, set them up in culture at a cell density of 3x106 cells/ml and challenge both the monocytes and PMN with endotoxin as in 1.1.

1.1a ALTERNATE PROTOCOL 1 (for rapid mononuclear cell gradients) TC "1.1a ALTERNATE PROTOCOL 1 (for rapid mononuclear cell gradients)" \l 3

Rather than using Percoll or Ficoll-Paque gradients to separate the mononuclear cells from the other leukocytes, one can use Lymphocyte Separation Medium (LSM; Organon-Technika), which seems to be useful across a wide array of species (e.g., human, bovine, equine, murine, ovine). The procedures employed are essentially identical to those for the Percoll gradients, with the exception that the cells are centrifuged on the gradients at 1500 rpm (i.e., 400xg) for 15 - 30 min. Thus the spin is shorter and at a much lower speed (which may be desirable in terms of the harshness of cellular treatment as well as in term so the times involved). The drawback to using an abbreviated centrifugation time is that neutrophils will not sediment to the bottom of the gradients in this time, but instead will remain suspended in and can be recovered from the LSM rather than as a pellet at the bottom of the tube.

Materials

all materials required for the Percoll isolation procedure (1.1)

Reagents

all reagents required for the Percoll isolation procedure (1.1)

Lymphocyte Separation Medium (LSM; Organon-Technika Inc)

METHOD

1-3.Obtain leukocyte/platelet-rich plasma as in the Percoll procedure (1.1).

4.Pipette 3 ml of LSM into a 15 ml p.p. conical centrifuge tube, then carefully overlay this with either the platelet-rich leukocyte-plasma or with platelet depleted, washed white blood cells.

5.Centrifuge the gradients for 15 - 25 min at 1500 rpm in the clinical centrifuge (i.e., 400xg).

6.Harvest the mononuclear cells as a band from the top of the density gradient medium, as in 1.1 - step 5, and wash them as in 1.1 - step 6.

1.1b ALTERNATE PROTOCOL 2 (fractionation of leukocyte sub-populations) TC "1.1b ALTERNATE PROTOCOL 2 (fractionation of leukocyte sub-populations)" \l 3

The individual leukocyte populations (i.e., monocytes, lymphocytes, basophils, eosinophils and neutrophils) can be at least partially purified as discrete bands directly from continuous density gradients. These are relatively easy to generate as custom gradients which can be tailored for individual needs by simply changing the low and high density "ends" of the gradient.

TABLE . Examples of useful density ranges for fractionating various cell populations

Materials

all materials required for the Percoll isolation procedure (1.1)

a continuous density gradient pouring apparatus (commercial or "home-made")

Percoll solutions of the required density

Reagents

all reagents required for the Percoll isolation procedure (1.1)

METHOD

1-3.Obtain leukocyte/platelet-rich plasma as in the Percoll procedure (1.1).

4.Pour the continuous density gradients by placing low density Percoll in the first chamber and high density Percoll in the second chamber. The medium is best delivered from the gradient pourer into the individual tubes using a peristaltic or other pump -- as the high density medium empties from the second chamber into the centrifuge tube, it is replaced and thereby diluted by low density medium. The second chamber should be equipped with a mixer or stirrer to ensure rapid and complete mixing of the reagents during this process. By steadily withdrawing the delivery needle as the centrifuge tube fills, you will generate a gradient of continuously decreasing density, with the density at the bottom of the gradient being equal to that of the medium in chamber 2 and that at the top of the gradient being equal to that of the medium in chamber 1.

5.When the gradient formation is complete, very carefully load the peripheral blood leukocytes to be fractionated on top of the gradients. Since the low density medium may be of a density very similar to that of the medium in which the cells are resuspended, great care may to be needed to avoid mixing of the cells and gradient medium.

6.Run the gradients for 25 min at 2000 rpm in the clinical centrifuge, just as in 1.1, and harvest them in an analogous manner. However, what you will notice with these gradients is that there are multiple bands, the precise number and their locations depending on the species and immune status of the blood donor. For example, quiescent eosinophils of humans and horses will run at a density equivalent to about _____g/ml, while activated eosinophils are hypodense, and will run as a discrete band at a density of about _____g/ml. Harvest each band into a separate tube.

7.Wash the cells from each band using an appropriate medium for your purposes (e.g., DMEM-10% or RPMI-10%) and count them using a hemocytometer. The cells can be morphologically identified on stained cytocentrifuge preparations.

1.2 Isolation of cells from the peritoneal cavities of mice TC "1.2 Isolation of cells from the peritoneal cavities of mice" \l 2 .

The serosal (abdominal or peritoneal) cavity of mice is a rich source of macrophages which are easily purified by simply flushing the cavity and performing a plastic adherence step (see 1.4). In this section, we will simply examine the method for lavage of the serosal cavity.

Materials

BALB/c mouse

anaesthetic XE "anaesthetic" (methoxyfluorane)

clinical centrifuge, 15 ml centrifuge tubes, 4 ml round bottom p.s. tubes

23-25 ga needles & 10 ml syringes

pipettes/pipettors (micropipettes and macropipettes)

sterile surgical tools (optional, for open body cardiac puncture)

laminar flow hood

inverted microscope

hemocytometer

Reagents

Ca++Mg++-free-HBSS (HBSS)DMEM-10% FCS (see Appendix A)

70% ethanol

cytocentrifuge

METHOD

1.Euthanize mouse with methoxyfluorane and cervical dislocation, and then wet its abdomen with 70% ethanol. Fully reflect the abdominal skin dorsally and ventrally, being careful to not tear any holes in the abdominal wall (small holes discovered during step 2 can be clamped off using hemostats).

2.Using a 25 ga needle, inject 10 ml of HBSS into the peritoneal cavity, through the abdominal wall, and then massage the distended gut to wash the serosal cells into the HBSS.

3.Slowly withdraw the fluid using a 23 ga needle on a 10 ml syringe and transfer the wash solution to a 50 ml polypropylene tube.

4.Repeat the wash with another 10 ml of HBSS and pool the two washings.

5.Wash the cells by centrifugation and resuspend to the desired cell density, in the desired medium. Determine the cell numbers using a hemocytometer.

1.3 Adherence purification of monocytes TC "1.3 Adherence purification of monocytes" \l 2 or macrophages

Monocytes or macrophages can be easily purified by taking advantage of the fact that they adhere rapidly and tenaciously to plastic (polystyrene, but not polypropylene) surfaces. So in this procedure, you will simply take the total mononuclear cell fraction from PBL as well as the total peritoneal lavage population and place the cells into the wells of multi-well microscope slides, a particularly convenient format for the experiments we will be doing. Macrophage adherence ostensibly works much better in the absence of protein (e.g., FCS), so this protocol will be performed using DMEM-0% FCS.

Materials/Reagents

all materials for purification of mononuclear and PMN from mouse PBL ( 1.1) or peritoneal macrophages (1.2)

plastic petri dishes or multi-well microscope slides

DMEM-0% FCS

DMEM-10% FCS

METHOD1.Resuspend the mononuclear or serosal cells at 3x106 cells/ml in DMEM-0% FCS and add 300 l of the cell suspension to each well of the multiwell slide.

2.Incubate the cells at 37C in the CO2 incubator for 3 h.

3.Remove the non-adherent cells by repeatedly pipetting the culture medium directly onto the cell monolayer that has formed on the bottom of each well. If you are not thorough enough at this stage, large numbers of lymphocytes will remain with the monocytes/macrophages on the plastic/glass surface. Remove the resuspended, non-adherent cells by aspirating the medium (save the aspirates if you want to retain the monocyte-depleted lymphocyte preparation). Repeat this procedure as needed, monitoring the success of washing by direct visual observation under the inverted microscope.

4.To remove the cells from the plastic, you can either remove the medium from the cells and replace it with 0.02% EDTA in saline, and then transfer the dishes onto ice for 10-15 min. By smoothly scraping the bottom of the dish with a cell scraper, 90 - 95% of the adherent cells will be dislodged as viable cells - wash the cells and resuspend in DMEM-10% FCS. Alternately, the cells can be removed by treating with EDTA and trypsin (this eliminates the need for scraping, but increases the wear and tear on the macrophage surface proteins).

5.To activate the cells in situ, just add LPS to a final concentration of 1-10 g/ml.

1.4 Antibody- or C3b-dependent phagocytosis XE "Antibody- or C3b-dependent phagocytosis"

TC "1.4 .i.Antibody- or C3b-dependent phagocytosis" \l 2 ;by macrophages;;

In this assay we will explore the potentials for macrophages to specifically interact with cells that have been targeted by the humoral immune system. That is, antibody-coated cells XE "antibody-coated cells" can be phagocytosed or otherwise killed by macrophages that bind the target via the FcR. We will use the classical example of opsinized (i.e., antibody- [or C'-] coated) sheep red blood cell phagocytosis by purified PBL monocytes. This is the basis for antibody-dependent cellular cytotoxicity XE "antibody-dependent cellular cytotoxicity" (ADCC) by immunoinflammatory cells against multiple types of cellular targets (e.g., tumour cells, bacterial cells, etc...).

Materialsmonolayers of PBL monocytes in 8-well multi-well slides ( 1.3); or use peritoneal lavage cells from normal or proteose/peptone-injected mice

humidified 37C CO2 incubator

clinical centrifuge & tubes

sterile eppendorf tubes

ReagentsC3b-coated zymosan beads (5.4.12)

0.5% suspension of SRBC in PBS

anti-SRBC-coated SRBC (5.4.13).

normal mouse serum (heat-inactivated at 56C for 30 min)

DMEM-10% FCS

METHOD1.Take the monocyte/macrophage monolayers XE "monocyte/macrophage monolayers" , in multi-well slides out of the 37C incubator and to pairs of the wells add either 20 l of either: the antibody-coated SRBC, normal SRBC suspension, C3b-coated zymosan, or 'activated' zymosan suspension. Incubate the cells/slides for 60 min at 37C.

2.Remove the slides from the incubator and wash the free SRBC or zymosan from the cultures by resuspending the sedimented cells with your pipetter and aspirating the contents. Examine the cells using the phase contrast condenser of the inverted microscope to determine the extent to which the SRBC/zymosan particles have been internalized by the macrophages (i.e., are very dull by phase contrast) or remain in the extracellular compartment (i.e., are very bright by phase contrast). Return the cells to the 37C incubator and periodically check them again to follow the internalization of the particles.

3.At the end of the experiment, take the well casing from the slide itself and fix the cells by immersion in 100% ethanol for 2 min. Allow the slides to air dry, and stain the slides with Giemsa stain (Appendix D).

5.Calculate the mean numbers (+/- SEM) of SRBC or zymosan particles in the macrophages in each treatment group. To do this, you will count the numbers of SRBC contained within 25 macrophages in each of ten 40x microscope fields, for each well on the slide. Perform an ANOVA test to determine the statistical significance of your results, and plot the data (including means, SEM, and probability values) on a graph.

1.5 Activation of macrophages XE "Activation of macrophages"

TC "1.5 .i.Activation of macrophages" \l 2 with bacterial lipopolysaccharide XE "lipopolysaccharide"

Monocytes or macrophages can be activated by the addition of many different kinds of reagents (e.g., immune complexes, interferon-, bacterial products, etc..). In this protocol, we will use the bacterial cell wall product lipopolysaccharide XE "lipopolysaccharide " (LPS XE "LPS" or bacterial endotoxin) to activate cultures of Pu5-1.8 (Pu5) cells, a murine macrophage cell line; Pu5 cells were one of the original lines used in the cloning of murine TNF. While we will be using this cell line (in order to save the lives of a number of mice), precisely the same protocol and approximately the same results would be obtained if we were to use freshly purified monocytes or macrophages. Later in the week we will then determine the extent to which you have activated the cells by quantifying their secretion of a number of monokines (i.e., IL-1, IL-6, and TNF).

Materials

laminar flow hoodhumidified 37C CO2 incubator

subconfluent monolayer cultures of Pu5-1.8 cells in DMEM-10% FCS

clinical centrifuge & tubes

inverted microscope

sterile eppendorf tubes

cell scraper

-20C freezer & freezer bags

Reagentsbacterial endotoxin, 1 mg/ml DMEM-0% FCS (E. coli lipopolysaccharide; LPS)

DMEM-10% FCS (see Appendix A)

METHOD

1.Take a look at the growing Pu5 cells under the inverted microscope to get a feeling of how they 'should' look under normal conditions.

2.Using a cell scraper, dislodge the Pu5 cells from the plastic and then transfer them to a 50 ml centrifuge tube. Remove 20 l of the cell suspension for cell counting with the hemocytometer.

3.While counting the Pu5 cells, sediment them by centrifugation (10 min at 1500 rpm in the clinical centrifuge). To resuspend them in fresh DMEM-10% FCS, completely aspirate the supernatant from the tubes, and then very briskly and repeatedly flick the tube with the cell pellet to disperse the pellet. Dispersal will be complete when the pellet has become a paste on the walls of the tube. Add sufficient DMEM-10% FCS to bring the cells to a concentration of 3x106 cells/ml, and then dispense the cell suspension into the wells of 24-well plates, at 1 ml per well. You will need 8 wells of cells (4 wells of unstimulated cells & 4 wells of stimulated cells) for todays experiment, as well as 4 wells which contain medium but no cells (LPS-medium controls)

4.Add LPS (to a final concentration of 10 g/ml) to the 4 'stimulated cells' wells and to the 4 'LPS-medium control' wells, and an equivalent amount of DMEM to the 4 wells of unstimulated Pu5 cells. Return the cultures to the 37C CO2 incubator.

5.Label the eppendorf tubes with your initials, the date, and the sample information (e.g., Pu5 supn't. + [or -] LPS; 1 [or 6, or 24] h). In addition to the supernatants from the cell cultures, save several aliquots of the DMEM-10% FCS that was used for the cultures (as medium controls)

6.At 1, 6, and 24 h post-challenge, examine the cells to get a feeling for whether the LPS has affected them in any visible manner. At each time, also harvest the culture medium from the appropriate wells, centrifuge them for a few minutes at full speed in the microfuge, and then aliquot each one into four eppendorf tubes (200 l/tube). Store all aliquots in the -20 freezer (long-term storage requires a -80 freezer).

7.At the end of the experiment, place all of the plasticware & cells in the contaminated-discard pan.

1.6 Monokine Bioassays XE "Monokine Bioassays"

TC "1.6 .i.Monokine Bioassays" \l 2 :;1.6.1 Assay for IL-1 activity XE "IL-1 activity"

TC "1.6.1 Assay for .i.IL-1 activity" \l 3 ;;

In this assay, we will depend on the property of LM-1 cells (a sub-clone of the ATCC cell line D10.G4) to proliferate in the presence of IL-1. While D10.G4 cells, like thymocytes, proliferate in response to IL-1 in the presence of sub-mitogenic doses of PHA or ConA, LM-1 cells do not need the PHA or ConA to respond to IL-1. Before the assay, the cells are rendered more sensitive to IL-1 by starving them of this cytokine for 5-7 days. The cell proliferation will be measured by examining the abilities of the cells in each well of the 96-well plates to take up and reduce the dye MTT to an insoluble blue-black formazan precipitate within their mitochondria. Thus the assays measures the mitochondrial activity of the cells, not the numbers of cells. While this is a very convenient parameter, it carries with it some problems, such as the influence agents that affect mitochondrial activity can have on the results.

Materialshumidified CO2 incubator

96-well tissue culture plates

micropipetters, tips

multi-channel pipetter

clinical centrifuge, tubes 15 ml

15 ml centrifuge tubes

hemocytometer

ELISA plate reader (with a 595 nm wavelength filter)

ReagentsLM-1 cells XE "LM-1 cells" which have not been fed fresh IL-1-containing medium for 5-7 days, and which were washed and held overnight in Click's-10% FCS without conditioned medium (i.e., IL-1-starved LM-1 cells)

Click's-10% FCS without conditioned medium (Click's-10% FCS-CM-)

recombinant mouse IL-1 (our present stock soln is at a concentration of 7.5 ng/l)

MTT XE "MTT" , (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), 5 mg/ml in PBS (stable for 2-3 wks at 4C)

acidified isopropanol (see Appendix C)

METHOD1. Starve the LM-1 cells 4-7 days before assay (feed them fresh conditioned medium some 4-7 days before the assay, and then do not feed them again). The night before the assay, wash the cells in fresh Click's-10% FCS-CM- and leave them in culture overnight.

2.On the day of the assay, resuspend the LM-1 cells to 4X105 cells/ml in Click's-10% FCS-CM- and dispense into the 96-well plates at 100 l/well, using a template pattern similar to the one depicted.

-Do not add any cells to the first column (i.e., wells A1, B1, ..., H1) of each plate, but do add all other reagents (e.g., Click's without IL-1) -- this will be the plate blank.

-Do not use the outer wells of the plate for any samples (there is an 'edge-effect' of the plates), but add DMEM-0% FCS to these wells.

-Always include a medium-only control (i.e., the same medium as that used for the experimental treatments, but which has not been exposed to the experimental cells). Use the same volume of experimental medium as that used in the test samples wells -- if you have multiple doses of test samples, use equivalent multiple doses of medium controls.

-In this format, one plate will accept 10 experimental samples, each done in quadruplicate.

3. Add 80 l of Click's-10% FCS-CM-, and 20 l of appropriately diluted rmIL-1 standard (like all samples and standards, in quadruplicate sets of wells) to a series of wells to achieve final doses of 0.5, 5.0, 50 and 500 pg/ml (see Appendix D, Generation of standard curves).

4. In parallel sets of quadruplicate wells, run samples (LPS-stimulated Pu5 cell culture supernatants) and medium controls (DMEM-10% FCS + 10 g/ml LPS) at required volumes (usually 5-20 l/well). Bring the final volume of each well to 200 l with Click's-10% FCS-CM-, and return the cells to the 37C humidified CO2 incubator for 3 - 5 days.

5. To measure the extent of the IL-1-driven LM-1 cell proliferation in each well, use the micropipetter to add 20 l of MTT stock solution (5 mg/ml) to each well, and then return the plates to the humidified 37C CO2 incubator for 1 - 2 h.

6.(Optional: centrifuge the plates for 10 min at 1500 rpm to sediment cells.) Carefully remove 150 l of medium from each well in the plate, and add 100 l of acidified isopropanol to each well. Agitate the plates on the ELISA plate shaker for 3 minutes, and then read the plates on the ELISA plate reader, set at a reading wavelength of 595 nm. Download the data to a 3-1/2" Macintosh-formatted floppy disc.

7.Use the Microplate manager program to crunch your data, and Statview+ to perform the statistical analyses. Plot your results as a bar graph (+/- SEM) using the Cricket III program provided.

1.6.2 Assay for IL-6 activity XE "IL-6 activity"

TC "1.6.2 Assay for .i.IL-6 activity" \l 3 ;;

Like the IL-1 assay, the IL-6 assay depends on the fact that 7TD1 cells proliferate strongly in response to low concentrations of IL-6. Again, we will measure this response using the MTT dye method.

Materialshumidified CO2 incubator

96-well tissue culture plates

micropipetters, tips

multi-channel pipetter

clinical centrifuge, tubes 15 ml

15 ml centrifuge tubes

hemocytometer

ELISA plate reader (with a 595 nm wavelength filter)

Reagents7TD1 cells in RPMI-10% FCS supplemented with rhIL-6 (80 pg/ml) XE "7TD1 cells in RPMI-10% FCS supplemented with rhIL-6 (80 pg/ml) " RPMI-10% FCS (see Appendix B)

recombinant human IL-6 XE "recombinant human IL-6" (rhIL-6; our stock solution is at 100 ng/ml)

MTT (5 mg/ml in PBS)

acidified isopropanol

METHOD1. Wash the 7TD1 cells two times in RPMI-10% FCS, and resuspend to a concentration of 2.5x104 cells/ml in RPMI-10% FCS.

2.Add 100 l of cells to each well in plate, using the same or a similar sample plate format to that indicated in 1.5.1 (IL-1 assay).

3.Add cytokine standards (2.5, 25, 250 & 2500 pg/ml final concentration) in 20 l of RPMI-10% FCS, and add your samples and experimental medium controls in appropriate volumes. In this assay, we will use 1.0, 2.0, and 5.0 l of the LPS-stimulated Pu5 cell culture supernatants, so you will need three sets of 'medium controls', each containing 1.0, 2.0 or 5.0 l of DMEM-10% FCS supplemented with 10 g/ml of LPS.

4. Return the plates to the 37C CO2 incubator for 3 days.

5. After 3 days, add 20 ul of MTT solution (5 mg/ml) to each well (including plate blank wells), and return plates to the incubator for 1-2 hr. When the mitochondria in each of the cells are plainly visible at 40x magnification, centrifuge the plates and remove 150 l of medium from each well. Add 100 l of acidified isopropanol (lysis buffer) to each well, vortex vigorously in the ELISA plate shaker for 3 min, and then leave the plates on your bench overnight.

6.The next morning, read the plates at 595 nm wavelength on the ELISA plate reader and download the data to a 3-1/2" Macintosh-formatted floppy disc.

7.Use the Microplate manager program to crunch your data, and Statview to perform the statistical analyses. Plot your results as a bar graph (+/- SEM) using the Cricket III program provided.

1.6.3 Cytotoxicity assay for TNF XE "Cytotoxicity assay for TNF"

TC "1.6.3 .i.Cytotoxicity assay for TNF" \l 3 bioactivity ;

TNF activity is usually detected using a cytotoxicity assay. L929 cells (or at least many of its sub-lines) are sensitive to TNF such that this cytokine kills the cells over 18 h. However, the cytotoxic effects of this cytokine are markedly diminished by the presence of other proteins (e.g., FCS), so that as much as possible, the assay should be run the absence of other proteins. Finally, L929 cells are most sensitive to the effects of TNF when the assay is run in the presence of a low levels of a transcription inhibitor (e.g., actinomycin D).Materialshumidified CO2 incubator

96-well tissue culture plates

micropipetters, tips

multi-channel pipetter

clinical centrifuge, tubes 15 ml

15 ml centrifuge tubes

hemocytometer

ELISA plate reader (with a 595 nm wavelength filter)

ReagentsL-929 cells XE "L-929 cells" in DMEM-10% normal horse serum (NHS).

DMEM-0% NHS

recombinant murine TNF standards XE "TNF standards" (diluted as in Appendix D)Actinomycin-D XE "Actinomycin-D" (stock 5 mg/ml in 95% ethanol)MTT (5 mg/ml PBS)

Acidified isopropanol (lysis buffer)METHOD1.On the day before the assay, harvest L929 cells from a flask by trypsinization (remove DMEM-10 % NHS medium and add DMEM-0% NHS medium. Add 4 - 5 drops of 1% trypsin/10 ml of medium and allow the typsin to digest the cells off of the plastic. This process can be expedited by watching the cells and, when they are beginning to lift well, but many still remain attached, forcefully slapping the flask down on your thigh. All cells will be dislodged instantaneously.

2.Wash the dislodged cells in DMEM-10% NHS medium, resuspend them to 4.5x105 cells/ml, and dispense 70 l to each well of a 96-well plate (for plate format, see 1.5.1; IL-1 assay). Return plate to the 37C CO2 incubator.

3.The next day, just prior to running the assay, remove all of the serum-containing medium from the wells by inverting the plate and vigorously flicking it. Add 180 l of DMEM-0% NHS containing 2.5 g/ml of actinomycin D (i.e., a 2000-fold dilution of the stock Act D).

4.Add TNF standards (0.04, 0.4, 4.0 and 40 units/well), control medium, and experimental samples (LPS-stimulated Pu5 cell supernatants & controls) to their appropriate wells, each in a 20 l total volume, so that the total well volumes equal 200 l.

5.Return plate to the 37C CO2 incubator overnight.

6. The next day, examine the cells under the inverted microscope to get a feeling for the relative levels of L929 cell death in each well. Add 20 l of MTT (5 mg/ml PBS) to each well (including plate blank wells), and again return the plates to the incubator.

7.After 45 - 60', re-examine the cells to confirm that adequate levels of MTT conversion to formazan dye have occurred in the mitochondria (see 1.5.1, IL-1 assay) and then remove 150 l of medium from each well in the plate and replace it with 100 l of acidified isopropanol.

8.Vortex the plates on an ELISA plate shaker to solubilize the formazan dye, and read the plates on the ELISA plate reader at 595 nm wavelength. Calculate the cytotoxicity in each of the wells using the formula:

percent cytotoxicity =

mean OD590 medium control wells - OD590 experimental well x 100

mean OD590 medium control wells

9.Calculate the mean (+/- SEM) cytotoxicities for the standards and each treatment group, express them in terms of units (+/- SEM) of TNF activity XE "TNF activity" (a unit of TNF activity XE "unit of TNF activity" is that amount of cytokine required to kill 50% of the cells in the assay) and graph your results. Perform a statistical analysis to confirm that your results are meaningful.

1.7. Neutrophil chemotaxis assay XE "Neutrophil chemotaxis assay"

TC "1.7. .i.Neutrophil chemotaxis assay" \l 2

Neutrophils are attracted in large numbers into inflammatory foci (e.g., loci of C' cascade activation, bacterial infections), where they are important to the clearance of bacteria or other insults both by virtue of their abilities to phagocytose the offenders, as well as through their toxic secretory products. These cells have receptors for an array of chemoattractants, including bacterial products (e.g., formyl-methionyl-leucyl-phenylalanine; fMLP), complement split products (e.g., C3a, C5a), and an array of chemokines (e.g., IL-8 or CINC in rodents). For this exercise, we will purify neutrophils from the peripheral blood of mice as in 1.1, then use these cells to study the chemoattractant activities of fMLP and C3a/C5a (i.e., zymosan-activated serum;5.4.11 ), as represented in zymosan-activated sera (i.e., C3a, C5a)..

Materialshumidified CO2 incubator

microchemotaxis chamber (NeuroProbe Inc)

PVP-free (PVPF) polycarbonate cell migration filters (5 m pore size; Millipore or NeuroProbe)

micropipetters, tips

purified peripheral blood granulocytes

ReagentsDMEM-0% FCS media

Ca/Mg--HBSS

f-met-leu-phe bacterial tripeptide

zymosan-activated serum ( 5.4.11)

interleukin 8 (or CINC)

METHOD

1.Generate a neutrophil-rich population from the peripheral blood, using dextran sedimentation (or if your experiments require purified cells, percoll-purified neutrophils), adjusting the population to a final concentration of 2x106 cells/ml of HBSS.

2.Prepare the chemoattactants for use in the assay, allowing approximately 30 l of each chemoattractant dilution. Dilute the zymosan-activated serum (ZAS) to final concentrations of 1:10, 1:100; 1:1000, and 1:10000; the fMLP to final concentrations of 10-6, 10-7, 10-8, 10-9, and 10-10 M; and the recombinant IL-8 to concentrations ranging from 50 pg/ml to 1000 ng/ml. Putative chemoattractant-containing biological samples should be diluted to 1:10, 1:20, 1:40, 1:80 and 1:160, as first estimates with unknowns.

3.To the bottom chamber of each well (samples should be run in duplicate, if not quadruplicate) add sufficient chemoattractant to completely fill the wells (do not overfill them, as interwell smearing of chemoattractant may occur when placing the polycarbonate membrane is step 4 -- a very slight convex surface to the sample is ideal).

4.Place the PVPF-free Nucleopore membrane (shiny side up) on top of the wells, being careful not to smear the chemoattractants between wells. (PVP-containing membranes will not retain cells that migrate completely through the pores in the assay, allowing them to drop off the membranes into the bottom chambers after completely traversing the porous membranes - thus, a complete assessment of the chemotactic response with these membranes would require enumerating the cells associated with the membranes, as well as those in the lower chambers.)

5.Place the plastic gasket on top of the membrane, and the top half of the apparatus on top of the gasket and firmly screw the lug-nuts down, such the a tight seal is created in each well.

6.Add 50 l of the responder cell population to each well, being careful to not generate air bubbles which will create an air-lock on top of the membranes (and thereby exclude cells from the membranes). Put the tip of your pipette just at the surface of the Nucleopore membrane, without contacting it, and quickly expel the 50 l of chemoattractant - with a bit of practice, this will become easy for you.

7.Place the chambers in a plastic dish containing damp paper towels in the 37C CO2 incubator, allowing 90 min for the neutrophils to respond to the chemoattractants. Eosinophils also respond in this time frame (i.e., 90 min) while monocytes and lymphocytes will call for 2.5-4 hr incubation times to respond.

8.Upon completion of the incubation period, disassemble the apparatus and carefully clamp the ends of the membrane with "bull-dog" or other suitable clamps, then remove the cells which settled onto the upper surface of the membranes in each well by scraping the upper surface across a scraper (e.g., a fairly sharp-edged glass microscope slide clamped into a ring-stand clamp).

9.Allow the membrane to air-dry, then stain with a suitable staining solution (e.g., Diff-Quick). After air-drying again, the membranes can be dipped in xylene to "clear" them and mounted in "Permount" or other mounting medium on glass slides. Mount the membranes with the bottom side of the membranes upper-most, so that the cells which have migrated completely through the membranes are most apparent. The nuclei of the cells that are still within the membrane pores will be visible as dark blue or purple shapes within the otherwise clear-to-light purple pores.

10.Count the cells with have migrated completely through the membranes, as well as those within the pores

1.8. FcRI-dependent activation of mast cells TC "1.8. FcRI-dependent activation of mast cells" \l 2

Mast cells XE "Mast cells" are found in increased numbers at the host's interface with its environment (e.g., skin, airways, intestinal tract) and seem to subserve a number of obvious functions (e.g., allergen-reactivity), but also perhaps some less obvious ones. For this class, we will take advantage of the fact that they produce very high levels of some cytokines and use them as a positive control for some of our experiments. Cl.MC/C57.1 cells produce higher levels of IL-4 than purified, fully differentiated Th2 lymphocytes, and higher levels of TNF than LPS-stimulated macrophages.

Mast cells can be activated by cross-linking (i.e., bridging) adjacent FcR-bound IgE molecules on the cell surface, so in these experiments we will sensitize some Cl.MC/C57.1 cells with a monoclonal IgE anti-DNP antibody XE "IgE anti-DNP antibody" , and then challenge with DNP-conjugated human serum albumin (DNP30-40HSA). The 2 hour supernatants from these cells will contain abundant TNF, and the cells will contain very high levels of TNF mRNA.

Materialshumidified CO2 incubator

T75 flasks

hemocytometer

micropipetters, tips

clinical centrifuge, tubes 15 ml

polypropylene 4 ml culture tubes

ReagentsDMEM-10% FCS media

Cl.MC/C57.1 cells in DMEM-10% FCS.

MAb IgE anti-DNP (ascites fluid; use at 1:3000 for Cl.MC/C57.1 cells).

DNP30-40HSA(stock solution, 1 mg/ml; use at 50 to 100 ng/ml).

METHOD

1.Obtain some Cl.MC/C57.1 cells and adjust the cell concentration to 3 X 106 cells/ml. You will only need 0.5 ml of mast cell supernatant/time point, so set up 3 ml of cells (i.e., 9x106 cells in 3 ml).

2.Add MAb IgE anti-DNP to the tube of C57 cells to a final IgE dilution of 1:3000, and incubate the cells for 30 - 60' at room temperature in order to saturate the cells' high affinity IgE receptors.

3.Wash the cells two times with DMEM-10% FCS and resuspend them again at 3x106 cells/ml. Bring the cells to 37C by placing them in a water bath. Add DNP30-40HSA to the cells to a final concentration of 10 ng/ml and place the tubes upright and lightly capped in the 37C CO2 incubator for 15 - 20 min to allow them to gas (i.e., exchange CO2 into the tube). After this, cap the tubes tightly and lay them on their side in the incubator (or use a slowly moving rotator at 37C).

4.At each of the indicated times after allergen challenge (i.e., 0.5, 1, 2, and 4 h), remove a tube of cells from the CO2 incubator and sediment the cells by centrifugation (8-10' @ 1500 rpm), and then aliquot the supernatants into labelled eppendorf tubes (100 l/tube). Freeze the aliquots at -20C (or -80C for longer term storage). The cells can be either discarded (as in our first Cl.MC/C57.1 experiment), fixed (e.g., for our in situ hybridization experiments), or processed for total cellular RNA extraction (e.g., our Northern analysis experiments).

2.0 ANTIBODIES: PURIFICATION & CHARACTERIZATION: TC "2.0 ANTIBODIES: PURIFICATION & CHARACTERIZATION:" \l 1 \n

In the second week of the course, we will grow up anti-mouse CD4 and anti-mouse CD8 monoclonal antibody-producing hybridoma cell lines, purify the IgG antibodies from the anti-CD4 cell culture supernatants by affinity chromatography, and analyse the purified antibodies and total culture supernatants by polyacrylamide gel electrophoresis and Western blotting. We will then functionally characterize the antibodies by using them for negative selection of CD4 and CD8 cells from mouse splenocyte populations. We will follow the success of depleting each of these populations using two-colour fluorescence activated cell sorter (FACS) analysis of the spleen cells.

2.1 Hybridoma cell culture with production of monoclonal antibodies XE "monoclonal antibodies"

TC "2.1 Hybridoma cell culture with production of .i.monoclonal antibodies" \l 2 Materials

T75 tissue culture flasks

clinical centrifuge and 50 ml polypropylene tubes

ReagentsGK1.5 (anti-CD4) XE "GK1.5 (anti-CD4)" and TIB 211 (anti-CD8) XE "TIB 211 (anti-CD8)" hybridoma cells

RPMI-10% FCS

METHOD1.Set up two T75 (i.e., 75 cm2) tissue culture flasks, one each for the TIB 211 and GK1.5 cells. Start each flask as a 40 ml culture, at a cell density of 105 cells/ml of RPMI-5% FCS medium.

2.Allow the cells to continue growing until the medium has gone completely yellow (acidic) and the cells have essentially all died (about 5 - 7 days; terminal cultures).

3.Collect the supernatant from each flask and centrifuge it for 15 min at 12,000 rpm in the RC-5B superspeed centrifuge to sediment all particulate matter.

4.Aliquot the supernatants and either process expeditiously or store at -20C or -80C (for longer-term storage).

2.2 Affinity purification of IgG antibodies TC "2.2 Affinity purification of IgG antibodies" \l 2 2.2.1 Avid-AL affinity chromatography TC "2.2.1 Avid-AL affinity chromatography" \l 3

AVID-AL is a popular new matrix with a natural affinity for IgG from a wide array of species, including rat. Thus, we will use it to purify the rat anti-mouse CD4 IgG antibodies from the GK1.5 hybridoma supernatants. Briefly, we will pour mini-columns of the Avid-AL matrix and run the hybridoma supernatants over the columns, thereby binding the IgG antibodies to the matrix. Following a washing step to remove the non-specifically bound proteins, we will elute the IgG using a low pH buffer, neutralize the eluate, dialyse it against PBS and concentrate it if necessary.

Materials

T75 tissue culture flasks

clinical centrifuge and 50 ml polypropylene tubes

AVID-AL IgG affinity column matrix XE "AVID-AL IgG affinity column matrix" 10 ml polyprep column, or 5-10 ml syringe and glass wool

dialysis tubing

centrifugal concentrators (e.g., Centricon tubes)

Reagents

AVID-CHROM Ig pure kit for purification of IgG

-column binding buffer (>1M proprietary salt)

-neutral elution buffer (1M Tris [pH 7.4], 20% glycerol)

-regeneration buffer (buffered methanol)

GK1.5 (anti-CD4 hybridoma) cells in RPMI-10% FCS

PBS

METHOD1.Collect the supernatant from a 4 day culture of GK1.5 cells (see 2.1) and centrifuge it for 15 min at 2,500 rpm in clinical centrifuge to sediment all particulate matter, and then filter the supernatant through a 0.45 m filter. pH the supernatant to 7.2 - 7.4.

2.Set up the affinity matrix column by pipetting 1.5 ml of a 50% matrix slurry into a 15 ml centrifuge tube and add 10 ml of regeneration buffer (methanol). Shake the tube vigorously to disperse the matrix and then sediment the matrix by centrifugation. Resuspend the matrix in 5 ml of binding buffer and pour this into a polyprep column. Wash the column with 10 ml of binding buffer.

3.Dilute the hybridoma culture supernatant fluid with 2 volumes of binding buffer (to bring the final salt concentration to 500 mM), and then run the supernatant through the affinity column matrix several times to saturate the IgG binding capacity of the matrix.

4.Wash the matrix with 15 - 20 ml of binding buffer, or until the phenol red from the culture supernatant fluid has leached from the matrix (it will turn from a brown to the lime-green colour).

5.Elute the IgG from the column by running 4 - 5 ml of the neutral elution buffer (1 M Tris [pH 7.5], 20% glycerol) through the column, collecting the eluate into one tube. Regenerate the column with 10 ml of regeneration buffer, and store in either binding salt (very short term storage; salts will precipitate with long term storage) or PBS (for longer term storage).

6.Dialyse the elute IgG overnight against three changes of PBS (1 liter ea.). After dialysis, determine the protein concentration of the eluted IgG solution using a Coomassie Brilliant Blue (AKA Bio-Rad or Bradford) protein assay (Appendix D) and, if necessary, concentrate the eluted protein using a centrifugal concentrator.

2.2.2 Protein A-Sepharose affinity chromatography TC "2.2.2 Protein A-Sepharose affinity chromatography" \l 3

Protein A from Staphylococcus aureus (Cowan strain) is a molecule with a very high affinity for IgG antibodies, and has been used for several decades as the protein of choice to purify these antibodies. We will use recombinant protein A bound to dextran beads (Sephadex-G50), but in the past people have used the bacteria themselves (fixed, of course) as affinity matrices for this purpose. In general, IgG1 antibodies will only bind to protein A at pH 8.0, while IgG2a and other IgGs will do so a more physiological pH (i.e., pH 7.2) as well as pH 8.0. The elution pH optima of IgG from the protein A columns is isotype-specific, with IgG1 elution being best performed at pH 6.5, IgG2a at pH 4.5, and IgG3 at pH 3.0 (the least hostile or acidic elution conditions available should be employed to prevent acidic hydrolysis of the antibodies)Materialsprotein A-Sepharose (Pharmacia, or Sigma)

clinical centrifuge and 50 ml polypropylene tubes

10 ml polyprep column, or 5-10 ml syringe and glass wool

spectrophotometer or UV monitor (equipped for reading OD 260)

dialysis tubing

Reagents

0.1 M citric acid (pH 4.5)

supernatant from a terminal culture of GK1.5 (rat IgG2a anti-CD4 hybridoma) cells

PBS (pH 7.2)

PBS/20% ethanol

1M Tris (pH 9.0)

METHOD1.Filter the GK1.5 culture supernatant through a 0.45 m filter and pH it to 7.2 - 7.4.

2.Pipette 1 ml of the protein A-Sepharose 50% matrix slurry into the column and wash it with several column volumes of PBS.

3.Run the GK1.5 culture supernatant through the column matrix several times to saturate the IgG binding capacity of the protein A.

4.Wash the column with PBS until no more protein elutes from the column, using a spectrophotometer or UV monitor (OD 260) to confirm the end-point.

5.Elute the IgG from the column by running 10 ml of the 0.1 M citric acid elution buffer through the column, collecting the eluate into one milliliter fractions (i.e., 1 ml/tube). In order to minimize the time that the antibodies remain in an acidic environment, we will elute the column directly into 1 M Tris buffer (pH 9.0).

6.Determine the protein contents of the eluted fractions by either determining the OD260 of the fractions, or by use of a protein assay (e.g., Coomassie Brilliant Blue [a.k.a. Bio-Rad, Bradford or CBB] protein assay; Appendix D), and pool the protein-containing fractions.

7.Regenerate the column with 10 ml of PBS, and store the matrix in PBS/20% ethanol.

8.Dialyse the eluted IgG overnight against three changes of PBS (1 liter ea.). After dialysis, determine the protein concentration of the eluted IgG solution using a and, if necessary, concentrate the eluted protein using a centrifugal concentrator.

2.3 Preparation of IgM antibodies TC "2.3 Preparation of IgM antibodies" \l 2

IgM antibodies do not adhere well to most of the available immunoglobulin affinity matrices (e.g., protein A, T gel, Avid-Chrom), so alternate methods must be employed to prepare IgM antibodies. A number of methods are available, with the simplest true purification probably being achieved by size exclusion chromatography (IgM pentamers have a molecular mass of 750 kD, while IgG and albumin have molecular masses of 150 & 65 kD, respectively). In this session, we will simply enrich for IgM antibodies by differentially "salting out" the IgM protein with ammonium sulfate. High concentrations of ammonium sulfate will cause the proteins in a solution to differentially precipitate out from their solubilized state - at 30% ammonium sulfate saturation, most of the IgM antibodies will precipitate out of solution, while at 45% saturation most of the IgG isotypes and the residual IgM antibodies will precipitate out of solution.

Materials

beaker and stir bar

clinical centrifuge & tubes

dialysis tubing

0.45 m filters

pH meter and pH reagents

stir plate

syringe and 20 ga needle

Reagents

culture medium from a terminal culture of TIB211 (IgM anti-CD8) hybridoma cells

saturated ammonium sulfate solution

borate-buffered saline

Method1.As with the IgG purification, generate a 4-day TIB211 hybridoma culture supernatant, sediment the particulate matter by centrifugation, then filter and pH the supernatants as in 2.2.

2.Place the supernatant in a beaker on a stir plate and drip saturated ammonium sulfate into the supernatant, while constantly stirring, to a final ammonium sulfate concentration of 30% (i.e., 0.5 volumes of ammonium sulfate into 1 volume of antibody). Use a syringe and 20-23 ga. needle (as required) to drip the ammonium sulfate into the antibody culture supernatant, drop-by-drop.

3.After the 30% ammonium sulfate has precipitated the available proteins in the hybridoma supernatants, allow the stirring to continue for an additional 30 min, then sediment the precipitate by centrifugation (15 min at 2500 rpm).

4.Return the supernatant to the beaker and continue dripping ammonium sulfate into the supernatant to a final ammonium sulfate concentration of 45%, and then sediment that as in step 3.

5.Dissolve the precipitated proteins in borate-buffered saline, dialyse overnight versus an excess of borate-buffered saline, determine the protein concentration using a CBB assay and aliquot and freeze.

2.4 Analysis & characterization of immunoglobulins TC "2.4 Analysis & characterization of immunoglobulins" \l 2

In this section, we will take the anti-CD4 IgG that you have purified, as well as the TIB211 anti-CD8 IgM antibodies and characterize them according to their size (molecular mass) and reactivity with anti-IgG and -IgM antibodies. We will use polyacrylamide gel electrophoresis XE "polyacrylamide gel electrophoresis" (PAGE) for the former, and Western blotting for the latter.

2.4.1 Polyacrylamide gel electrophoresis of immunoglobulins TC "2.4.1 Polyacrylamide gel electrophoresis of immunoglobulins" \l 3

PAGE is a powerful technique for confirming the presence of proteins in solutions, although it is lacks the specificity of Western blots in identifying the proteins (beyond their molecular weights). However, it is an ideal method to confirm the homogeneity or lack thereof of purified proteins.

MaterialsPAGE mini-gel apparatus and power pack

Reagents

Commercial acrylamide/bisacrylamide solution (40% acryl/0.8% bisacryl)

Commercial separating and stacking gel buffers

ammonium persulfate XE "ammonium persulfate" , 10% solution (freshly prepared)

isobutyl alcohol (H2O-saturated)

PAGE 2x sample prep buffer (for denaturing & reducing the samples)

PAGE 5x SDS/run buffer (dilute 1:5 with H2O before use)

PAGE gel fix solution (25% isopropanol, 10% acetic acid)

rapid Coomassie blue stain XE "rapid Coomassie blue stain" (0.006% Coomassie Brilliant Blue G-250 in 10% glacial acetic acid)

protein samples

biotinylated molecular weight markers (for Western blots)

unstained molecular weight markers (for protein staining gels)

METHOD1.Clean the glass plates and gaskets, and assemble the PAGE apparatus. Place a mark on the glass at the 6 cm mark (from the bottom).

2.Mix together in a side-arm flask, 3.9 ml of the acrylamide/bisacrylamide solution, 3.75 ml of the separating gel buffer, and 7.2 ml of H2O. Degas the solution under vacuum for 10 - 15 min. Add 150 l of ammonium persulfate, and mix once again by swirling gently, and pour the acrylamide separating gel XE "separating gel " mixture into the PAGE apparatus up to the 6 cm mark, and then overlay the mixture with H2O-saturated isobutyl alcohol. XE "H2O-saturated isobutyl alcohol." Allow the PAGE gel reagents to polymerize, and then pour off the alcohol overlay and rinse the top of the gel with water.

3.In another side-arm flask, mix together 0.5 ml of the acrylamide/bisacrylamide solution, 4.5 ml of the stacking gel buffer, and degas the solution under vacuum for 10 - 15 min. Add 25 l of ammonium persulfate, and mix once again by swirling gently, and pipette this solution on top of the polymerized separation gel. Immediately place the well-forming comb in place, with the teeth submersed in the stacking gel XE "stacking gel" solution. Allow the stacking gel to polymerize, and then remove the comb from the top of the stacking gel.

4.Attach the gels/plates to the electrode frame and seal the seams with agarose. Place this assembly into the PAGE run reservoir and fill the reservoir and top of the gel assembly with PAGE run buffer.

5.Prepare samples for the PAGE run while the stacking gel is polymerizing. To do so, mix the protein sample 1:1 with sample prep buffer in an eppendorf tube and place in a boiling water bath for 5 min. Remember to run the appropriate molecular weight markers, . Pulse microfuge the tubes to sediment samples and hold on ice until running them on the gels. Carefully pipette each sample into a well of the gel. 6.Run the gel at 150 volts until the bromophenol blue dye XE "bromophenol blue dye" front reaches the bottom of the gel. Turn off the power, disassemble the PAGE gel apparatus.

7.Incubate the gel for 10 - 15 min in isopropanol fix XE "isopropanol fix" solution, stain it in the rapid Coomassie brilliant blue XE "rapid Coomassie brilliant blue" for 2 h to overnight at room temp, and then destain in several changes of 10% glacial acetic acid over 5-8 h.

8.For permanent storage, the gel can be dried down using a commercially available drying apparatus.2.4.2 Western blotting to detect immunoglobulins TC "2.4.2 Western blotting to detect immunoglobulins" \l 3 In Western blotting, proteins that have been fractionated on PAGE gels are electrophoretically transferred to nitrocellulose or other types of membranes (e.g., derivatized nylon), to which they may differentially bind, and then the protein bands on the membranes are visualized immunochemically, using alkaline phosphatase- or horse radish peroxidase-labelled antibodies and appropriate chromogens. Western blotting is a very sensitive method of detecting proteins to which antibodies already exist.

Materials

Western blotting transfer apparatus (wet)

Whatman #1 filter paper

nitrocellulose transfer membrane (do not handle with bare hands)

Reagents

PAGE gel with separated proteins to be transferred (unfixed)

Western blotting transfer buffer

TTBS (Tris-buffered saline with 0.1% Tween 20)

TTBS-5% Carnation skim milk powder

PBST (PBS with 0.05% Tween 20)

bromo-chloryl-indoyl phosphate/nitroblue tetrazolium (BCIP/NBT) substrate

streptavidin-alkaline phosphatase conjugate XE "streptavidin-alkaline phosphatase conjugate " (strep-AP; diluted 1:5000 in PBST)

METHOD1.Equilibrate the gel after electrophoresis to Western blot transfer buffer by incubation for 45 min. Also equilibrate a sheet of nitrocellulose XE "nitrocellulose" (cut to the same size as the half the gel to be used for Western blotting) to the same buffer.

2.Transfer the gel to the gel blotting bracket, such that the gel is sandwiched immediately next to the nitrocellulose (with no air bubbles between the two) and both are sandwiched between two sheets of filter paper, with two 'brillo' pads flanking the filter paper. This arrangement of gel, nitrocellulose membrane and pads (depicted below) is clamped between the Western blotting electrodes such that the gels is on the negative electrode side and the nitrocellulose is on the positive electrode side (the proteins will be negatively charged due to the SDS in the PAGE sample prep buffer).

3.Set the gel assembly in the gel apparatus, fill it with transfer buffer and then move the whole apparatus to the cold room. Run the transfer at 100 volts for 2-3 h (IgM may migrate through the nitrocellulose membrane in 3 h).

4.Disassemble the transfer assembly and stain the gel as in 2.4.1.

5.Transfer the nitrocellulose membrane into a large weigh boat containing 15 ml of TTBS-5% Carnation skim milk powder for 2 h to overnight to block the non-specific binding of other proteins to the nitrocellulose membrane.

6.Wash the membrane two times for 5 min each with PBST and place the blot into 15 ml PBST containing biotinylated rabbit anti-rat IgG and IgM (1:1500 final dilution) for 60 min at room temperature.

7.Wash the blot three times 5 min in PBST and place the blot in 15 ml of PBST containing a 1:5000 final dilution of streptavidin-alkaline phosphatase conjugate for 90 min at room temperature.

8.Wash the blot three times 15 min in H2O, and then transfer into 15 ml of freshly-prepared TMS -BCIP/NBT mixture. (To prepare the BCIP-NBT substrate: to 5 ml of 0.1 M Tris (pH 9.5), 0.1 M NaCl, 0.05 M MgCl2, add 22 l BCIP; mix by inverting then add 16.5 l NBT and again mix). Incubate for 30 - 40 min until the bands become well-defined, and then wash the blot with H2O and air dry.

9.Plot the relative migration distances of each of the molecular weight marker proteins on a graph, and use the plot and the migration distances of the bands in the IgG and IgM lanes to interpolate the relative molecular weights of the proteins detected, and thereby confirm their identities.3.0 T CELL AND B CELL RESPONSES: TC "3.0 T CELL AND B CELL RESPONSES:" \l 1 \n

3.1 C'-dependent depletion of CD4+ and CD8+ T Cells TC "3.1 C'-dependent depletion of CD4+ and CD8+ T Cells" \l 2

In this exercise, we will learn how to deplete selected populations of cells from a heterogeneous mixture of cell types. Specifically, we will use anti-CD4 or anti-CD8 antibodies and C'-dependent cytotoxicity XE "C'-dependent cytotoxicity" to deplete all of the CD4+ or CD8+ cells, respectively, in a single cell suspension of mouse splenocytes. We will confirm this depletion by using commercially available fluorescein-labelled anti-CD4 XE "fluorescein-labelled anti-CD4" and phycoerythrin-labelled anti-CD8 antibodies XE "phycoerythrin-labelled anti-CD8 antibodies" to stain the residual cell populations and then we will analyse the composite phenotype of these cells using two-colour FACS XE "two-colour FACS" (fluorescence-activated cell sorter) techniques.

Materialssingle cell suspension of mouse splenocytes (at 1x107 cells/ml; Appendix D)

clinical centrifuge and 4, 15 and 50 ml polypropylene tubes

ReagentsGK1.5 (anti-CD4 hybridoma) cell supernatants (from a 2-4 day culture)

TIB211 (anti-CD8 hybridoma) cell supernatants (from a 2-4 day culture)

purified GK1.5 IgG antibodies

purified HB121 IgG antibodies (control IgG; specificity: mouse IgG1 anti-human IgE)

fluorescein-labelled anti-mouse CD4 IgG antibodies

phycoerythrin-labelled anti-mouse CD8 IgG antibodies

Low-tox rabbit C' XE "Low-tox rabbit C' " (Cedar Lane)METHOD1.Label and set up a series of tubes to receive reagents as follows:

LABELCELLSANTIBODY (g/ml)COMPLEMENT (l)

no Ab, no C'10000; medium only

HB 121 (control)10010 100

GK1.5 supn't10010100

GK1.5 IgG10010100

1001.0100

1000.1100

TIB211 supn't10010100

TIB211 IgM (30% AS)10010100

1001.0100

1000.1100

TIB211 IgM (45% AS)10010100

1001.0100

1000.1100

2.Add 100 l of the splenocyte suspension (i.e., 106 cells) and 100 l of appropriately diluted antibody (10, 1.0 or 0.1 g protein from the HB121, GK1.5, or TIB211 preparations) to each tube. Incubate the cells with the antibody for 30 min at room temperature.

3.Add 100 l of the guinea pig complement to each tube, and incubate the tubes at 37C for 30 min. Wash the cells two times with DMEM-10% FCS (8 min at 1500 rpm in the clinical centrifuge).

4.Resuspend the cells to 100 l in DMEM-10% FCS, and add 3 l of FITC-labelled anti-CD4 and 3 l of PE-labelled anti-CD8 IgG to each tube and incubate the cells on ice for 30 min.

5.Wash the cells once more with an excess of DMEM-10% FCS and resuspend to 100 l with DMEM-10% FCS. Add 100 l of 1% paraformaldehyde XE "paraformaldehyde" and fix the cells on ice for 30 min, then wash the cells one time with PBS and resuspend to 100 l with PBS.

6.Store the cells in the refrigerator overnight, for FACS analysis the next day.

3.2 MACS purification (or depletion) of CD4+ and CD8+ T Cells TC "3.2 MACS purification (or depletion) of CD4+ and CD8+ T Cells" \l 2

In this complementary exercise, we will learn how to use antibody-coated paramagnetic beads that are specific for the isotypes of the anti-CD4 and anti-CD8 antibodies that you generated in 2.1 to purify all of the CD4+ or CD8+ cells, respectively, from a single cell suspension of mouse splenocytes. The real advantage of this system is that the selected populations are not killed or otherwise damaged by the isolation procedure, so that it may be possible to use them for functional studies after the purification procedure -- this is what we do in our lab to purify mast cells from tissues. The potential disadvantage is that in some cases the cell surface marker employed may well, by itself, activate or otherwise alter the physiology of the cells (e.g., anti-CD3 antibodies may activate T cells). We will confirm our depletion by using commercially available fluorescein-labelled anti-CD4 and phycoerythrin-labelled anti-CD8 antibodies to stain the residual cell populations and then we will analyse the composite phenotype of these cells using two-colour FACS (fluorescence-activated cell sorter) techniques.

Materialssingle cell suspension of mouse splenocytes (at 1x107 cells/ml; Appendix D)

clinical centrifuge and 4, 15 and 50 ml polypropylene tubes

Mini-MACS separation column (type MS; capacity 107 positive cells/column)

Mini-MACS column magnet (Miltenyi Biotec Gmbh)ReagentsGK1.5 (anti-CD4 hybridoma) cell supernatants, purified IgG (2.1)

TIB211 (anti-CD8 hybridoma) cell supernatants, ammonium sulfate ppt. IgM (2.1)

PBS (pH 7.2), containing 5% FCS & 2 mM EDTA (PBS/EDTA)

mouse anti-rat IgG-conjugated paramagnetic beads (Miltenyi Biotec)

mouse anti-rat IgM-conjugated paramagnetic beads (Miltenyi Biotec)

fluorescein-labelled anti-mouse CD4 IgG antibodies

phycoerythrin-labelled anti-mouse CD8 IgG antibodies

METHOD1.Label and set up a series of tubes to receive reagents as follows:

2.Add 1 ml of the splenocyte suspension (i.e., 107 cells) and 250 l of appropriately diluted antibody (HB121, GK1.5, or TIB211; i.e., 25 g) to each tube. Incubate the cells with the antibody for 30 min at room temperature.

3.Wash the cells one time with DMEM-10% FCS (8 min at 1500 rpm in the clinical centrifuge) and resuspend to 300 l of PBS/EDTA.

4.While the cells are washing, also wash the Mini-MACS column, flushing it through with the PBS/EDTA. If air bubbles are present in the column, it may be necessary to back-flush the columns to get rid of the air bubbles. Mount the column in the magnetic holder in preparation for the separation in step 7 below.

5.Add 25 l of the anti-IgG paramagnetic beads to the GK1.5 tube from step 2 (i.e., 1:40 beads:cells), 25 l of the anti-IgM beads to the TIB211 tube, and 25 l of each to the no antibody and HB121 tubes, and incubate for 30 min at 6-12C (ice-water bath).

6.Wash the cells in PBS/EDTA, resuspending them to 300 l PBS/EDTA.

7.Apply the washed cells to the column (in the magnetic holder), and collect the flow through, then wash the column two times with an additional 1 ml (each time) of PBS/EDTA, collecting the flow-through each time. Pool the flow through cells, which comprise the marker depleted populations, and wash and count them.

8.Remove the column from the magnet, clamping it to a ring stand, and flush the bound cells out of the column with 2 ml of PBS/EDTA, using the column plunger to assist in this operation, and collecting the eluted cells in a sterile tube..

9.Wash and count under the hemocytometer the retained cell populations. You will use these counts to compare with those obtained by FACS analysis of the total spleen populations (no Ab treatments).

8. Stain the unselected populations from step 6 with the FITC-anti-CD4 and PE-anti-CD8 antibodies at in 3.1, and fix with paraformaldehyde for FACS analysis the next day.

3.3 Assessment of T cell proliferation TC "3.3 Assessment of T cell proliferation" \l 2 (Blast assay XE "Blast assay" )

One of the basic tests in immunology is that of confirming that T cells are responding to antigens as they should, or determining the precise levels at which these cells respond to antigens (specific immunoreactivity of the cells) or mitogens (overall responsiveness of the T cells). Traditionally, immunologists set up the target T cell population (e.g., PBL mononuclear cells, lymph node or spleen cells) in tissue culture, challenged the cells with the antigen of interest and then measured the proliferation of the T cells three days later by ascertaining their uptake of a radiolabelled DNA precursor (e.g., the nucleotide 3H-thymidine). This is a very sensitive assay, but it calls for the use of equipment and facilities specialized for use in handling radioisotopes. More recently, many labs have been using the MTT assay, performed exactly as outlined above for the LM-1 (IL-1 ASSAY) and 7TD1 (IL-6 ASSAY) cell proliferation assays, to accomplish the same goal.

In this assay, we will examine the overall responsiveness of splenic T cells from BALB/c mice, by challenging the splenocytes with the T cell mitogen concanavalin A (Con A). In order to optimize the system however, we will need to determine the optimal concentrations of spleen cells needed for the assay, as well as the optimal doses of Con A required to induce proliferation. As with most immune responses, too much or too little of either can be inhibitory to the responses we wish to examine.

Materialshumidified CO2 incubator

96-well tissue culture plates

micropipetters, tips

multi-channel pipetter

clinical centrifuge, tubes 15 ml

15 ml centrifuge tubes

hemocytometer

ELISA plate reader (with a 595 nm wavelength filter)

ReagentsBALB/c mouse splenocytes (5x107 nucleated cells/ml) in DMEM-10% FCS

DMEM-10% FCS media

Concanavalin A XE "Concanavalin A" (our stock is a 4 mg/ml solution in DMEM)

MTT (5 mg/ml in PBS)

acidified isopropanol

METHOD1.In one 96-well plate, set up both cell number-response and Con A XE "Con A" dose-response curves; using the plate format indicated below, and the volumes indicated in the table.

CELL NUMBERS TITRATIONConA DOSE-RESPONSE CURVE

[CELL]

(x106/well)medium

(l)cells

(l)ConA

(l)[ConA]

(g/well)medium

(l)cells

(l)ConA

(l)

0.11782200.11305020

0.251755200.251305020

0.517010200.51305020

1.016020201.01305020

2.513050202.51305020

5.0801002051305020

7.53015020101305020

For the cell numbers titration portion of the experiment, use a ConA concentration of 2.5 g/ml. For the ConA dose-response curve, use a splenocyte concentration of 2.5x106 cells/well. Add sufficient DMEM-10% FCS to each well to bring the well volumes to 180 l, and then add 20 l of appropriately diluted ConA to each well (i.e., total well volume of 200 l).

2.Place the plates in the 37C CO2 incubator for three days.

3.At the end of the three days, examine the cells in each well to get a feeling for how the cells have responded to the ConA (strong ConA mitogenic responses will have induced cell clumping). Next, add 20 l of MTT solution (5 mg/ml) to each well and return the plates to the incubator for 45 - 90 min.

4.Remove 150 l of medium from each well, taking care not to also aspirate cells from the bottom of the wells. Add 100 l of acidified isopropanol to each well and place on the ELISA plate shaker for 3 - 4 min, and then read the plate at 595 nm wavelength on the ELISA plate reader.

5.Calculate the mean OD595 (+/- SEM) for each treatment group, perform the statistical analyses and plot your data using bar graphs.

3.4 Plaque Forming Cell (PFC) assay for IgM-producing cells TC "3.4 Plaque Forming Cell (PFC) assay for IgM-producing cells " \l 2 Materials

sheep red blood cells (SRBC; 2x108/ml), washed in PBS

20% (v/v) SRBC in PBS/10%FCS

syringes & 27 or 30 ga needles

mice

PFC assay chambers

methoxyfluorane (anaesthetic)

Reagents

PBS

H20 & 10x HBSS for hypotonic lysis of spleen RBC

guinea pig serum diluted 1:2 in PBS/10% FCS

Method

1.Inject 0.2 ml of the SRBC suspension (i.e., 4x107 cells) either intravenously or intraperitoneally into 8-10 week old mice.

2.Euthanize the mice after 4 days if they were immunized intravenously (or after 5 days if they were vaccinated intraperitoneally), and generate single cell suspensions from their spleens. Lyse the residual splenic red blood cells by hypotonic or ammonium chloride lysis (5.4.8) and bring the nucleated cells to a final concentration of 5x106 cells/ml

3.To a series of labelled eppendorf tubes, add either 0, 30, 60, or 120 l of the splenocyte suspension, 30 l of the 20% SRBC suspension (i.e., in PBS/10% FCS), 30 l of diluted guinea pig serum and 240, 210, 180, or 120 l of RPMI-10%, as appropriate to bring the final volume of each tube to 300 l, and thoroughly mix the contents of each tube.

4.Load 80 l of the mixture into each assay slide chamber (see diagram below) and seal each with wax.

5.Incubate the chambers for 1 - 1.5 h at 37C.

6.Remove the chambers from the incubator and count the numbers of plaques of red blood cell lysis under the microscope.

3.5 ELISPOT assays for single cytokine- or Ab-producing cells TC "3.5 ELISPOT assays for single cytokine- or Ab-producing cells" \l 2

A very powerful method to assess the abilities of animals to produce antibodies (Ab) or cytokines in response to antigenic stimulation is the ELISPOT assay. This procedure is used to detect the abilities of individual B or T (or other) cells to secrete their products. We will assess the abilities of splenocytes from BALB/c mice that have been immunized with ovalbumin-alum to produce ovalbumin-specific antibodies of varying isotypes or those of these mice or SRBC-vaccinated mice to produce IL-4 or IFN- in response to antigenic challenge in vitro. Thus, we will in effect by phenotyping the CD4+ T cell responses (i.e., Th1 or Th2) of the mice to these immunogens.

Materials

splenocyte suspensions from mice vaccinated with:

- ovalbumin/alum (i.p.: on dy 1 and dy 14; harvest cells on dy 21)

- SRBC (i.v.: dy 1, 200 l of 0.1% SRBC; dy 14, 200 l of 1% SRBC)

pipettes/pipettors

clinical centrifuge, 15 ml tubes

96-well ELISPOT XE "ELISPOT" plate

Reagents

anti-mouse IL-4 and anti-mouse IFN capture antibodies (1 g/ml coating buffer stock) XE "capture antibodies (1 g/ml coating buffer stock)" bromo-chloryl-indoyl phosphate/nitroblue tetrazolium (BCIP/NBT) substrate

biotinylated anti-mouse IL-4 and anti-mouse IFN antibodies (detection antibodies XE "detection antibodies" )

DMEM-10% FCS

ELISPOT blocking solution; (DMEM-10% FCS)

ELISPOT coating buffer; (carbonate/bicarbonate, pH 9.4)

Lymphocyte separation medium XE "Lymphocyte separation medium" ovalbumin (5 g/ml coating buffer stock solution)

PBST XE "PBST " (PBS with 0.05% Tween 20)

streptavidin-alkaline phosphatase conjugate XE "streptavidin-alkaline phosphatase conjugate " (strep-AP; diluted 1:5000 in PBST)

METHOD

1.Precoat each of the ELISPOT plates XE "ELISPOT plates" with capture antibodies or antigen (this can be done several days in advance). To do this, to each well of the plate, add the purified anti-IL-4 or anti-IFN antibodies in ELISPOT coating buffer at a concentration of 1 g/ml or the ovalbumin at a concentration of 5 g/ml. Cover and seal the plates with parafilm and incubate overnight at 4C.

2.The next morning, remove the capture antibody from the wells by inverting the plate and sharply flicking it. Rinse out each well with 200 l of blocking solution by pipetting the solution up and down several times with a multichannel pipetter (do not touch the bottom of the well with the pipette tips!). Remove the blocking solution, rinse as above and add 100 l of fresh blocking solution to each well. Incubate the plates at 37C for a minimum of 1 hour (or until the cells from the next step are ready).

3.Generate a single cell suspension from the spleens of an OVA- and SRBC-sensitized mice, and resuspend the cells to 1x107 cells/ml DMEM-10% FCS.

4.Remove the ELISPOT plate from the incubator and dump out the blocking solution. Add 100l of spleen cells and 100 l of antigen (ovalbumin @ 2.5 g/ml or SRBC @ 1.0% suspension) to each well, and incubate the plates at 37 C for 8 hours. Do not to disturb the plates while they are incubating, so that each cell secretes all of its cytokine/antibody in only one location.

5. After 8 h, remove the cells from the plate by first agitating the plates on the ELISA plate shaker for 3 min, and then inverting them and vigorously flicking the contents from the wells. Continue washing the wells with 200 l of PBST -- vigorously pipette the contents up and down, but do not touch the bottom of the wells, and flick the PBST out as above. Remove the excess PBST by banging the plate upside down on paper towels. Repeat this wash procedure 6 times.

6.Add 100 l of biotinylated anti-cytokine or anti-isotype antibody (diluted to 1 g/ml in PBST) to each well, and incubate the plates overnight at 4C.

7.Wash the plates 5 times with PBST as in step 6, add 100 l of diluted strep-AP to each well, and incubate the plates for 1.5 h at room temperature.

8.Wash the plates 10 times by repeatedly dunking the plate in a large beaker of distilled-deionized H2O, each time removing all of the H2O from each well as above (i.e., flicking and banging on paper towels). Careful washing is important, for it will reduce the assay background substantially.

9. Add 100 l of freshly prepared BCIP/NBT substrate to each well. (To 5 ml of 0.1 M Tris (pH 9.5), 0.1 M NaCl, 0.05 M MgCl2, add 22 l BCIP, mix by inverting and then add 16.5 l NBT and again mix; for smaller volumes, use 3.75 ml buffer, 16.5 l BCIP & 12.37 l NBT).

10.Incubate the plates at room temperature in the dark until spots develop or the plate

background begins to increase (approximately 30-45 minutes).

11.To stop the reaction, flick out the substrate and wash the plates 3 times in H2O by the dunking method. Allow them to dry overnight (with the lids off), as the background fades dramatically when the plates dry.

12.Count the spots under low magnification under the dissecting scope, or using an image analyser

3.6 ELISA ASSAY XE "ELISA ASSAY"

TC "3.6 .i.ELISA ASSAY" \l 2 S; (Enzyme-linked Immunosorbent Assay XE "Enzyme-linked Immunosorbent Assay" )

The ELISA assay is a powerful method for the detection of specific antigens (be they from pathogens or those specific for antibodies or cytokines). It can detect many antigens with a sensitivity of 1 pg/ml, although for many others sensitivities of 100-200 pg/ml are difficult to achieve. There is very little difference between the ELISPOT and ELISA assays - the ELISPOT detects cytokine or antibody production in situ by viable cells while the ELISA detects proteins which are present in a soluble form in biological fluids. The former assay is performed in nitrocellulose paper-lined 96-well plates and utilizes a precipitating indicator dye (so that individual cell traces are detected), while the latter is performed in high protein-binding plastic 96-well plates and employs soluble indicator dyes. While the ELISPOT tells you how many cells are secreting the antigen being detected, the ELISA will quantify the product precisely.

3.6.1 ELISA assay for detection of antigen-specific antibodies TC "3.6.1 ELISA assay for detection of antigen-specific antibodies" \l 3

In the antigen-specific antibody ELISA, the wells are coated with the non-antigen-specific immunoglobulin standards or the antigen in question, the latter should capture the antigen-specific antibodies from the samples. and then blocked. Subsequently, the wells are blocked with a protein solution that is not recognized by the capture antigen or standards (e.g., DMEM-10% FCS or 1% bovine serum albumin), and then the biological samples are applied. The captured antigen-specific antibodies are then detected precisely as are the cytokines/antibodies in the ELISPOT assay (3.5)

Materials

Immulon-4 ELISA plates

pipettes/pipettors

Reagents

experimental sera or other putative antibody source (e.g., from ova-vaccinated mice) ovalbumin for capture of antigen-specific antibodiesIgG1, IgG2a, and IgE standards (commercial or appropriate hybridoma supn't)

biotinylated IgG1, IgG2a, and IgE detection antibodiesELISA carbonate coating bufferPBST (PBS with 0.05% Tween 20)

DMEM-10% FCS

streptavidin-allkaline phosphatase (SA-AP) or SA-horse radish peroxidase (SA-HRP)

3 mM p-nitrophenyl phosphate (in 0.05 M Na2CO3/0.05 mM MgCl2; substrate for SA-AP)

2-2'-azino-di[3-ethyl-benzthiazoline sulfonate (6)] with H2O2 (ABTS;

1 component substrate for SA-HRP)

1% sodium dodecyl sulfate (SDS; optional stop solution for reactions)

METHOD

1.Coat the wells of the Immunolon-4 plates with the antigen to be used for antibody capture (ovalbumin @ 5 g/ml in carbonate coating buffer) or with the diluted antibody standards, as appropriate. Add 100 l per well, then cover the plate(s) and incubate overnight at 4C.

2.Wash the wells 2 times with PBST. To do this, one can either use a squirt bottle of PBST or a multichannel pipettor to fill each of the wells and allow to stand for 30 - 60 seconds, then turn the plate upside down and flick the wash fluid into a sink, then fairly forcefully hit the plate upside down on a stack of paper towels to remove the excess fluid from each well. Block the plates by adding 200 l of DMEM-10% FCS to each well and incubate, covered, at room temperature for 2 hours.

3.Wash the wells 2 times with PBST as in step 2, and to the ovalbumin-coated sample wells add 100 l of the samples (diluted 1:50 in DMEM-10% FCS) to each well. Add 200 l of PBST to the antibody standard wells. Cover the plate and incubate overnight at 4 C.

4.Wash the wells 4 times with PBST. Dilute the biotinylated anti-Ig isotype

antibodies to 0.5 - 2.5 g/ml in PBST (the optimal concentration will need to be determined empirically), and add 100 l of each per well, as appropriate for each sample or standard. Cover and incubate at room temperature for 90 min.

5.Wash the wells 8 - 10 times with PBST, and add 100 l of either SA-HRP (1:5000 in PBST) or SA-AP (1:5000 in PBST) to each well. Incubate at room temperature for 90 min.

6.Wash the plates 8 - 10 times with distilled H2O by the dunking method, making sure to remove the excess fluid as above. Add 100 l of the ABTS substrate (SA-HRP avidin-enzyme conjugate only) or freshly prepared 3 mMp-nitrophenyl phosphate substrate (SA-AP avidin-enzyme conjugate only) to each well, then place the plates in a dark location (e.g., a drawer works well) and allow the reactions to develop for 20 - 45 min at room temperature. The plates can be read directly at this point, or stop solution may be added to reduce plate to plate variability when reading multiple plates. For the ABTS substrate, the stop the reactions by adding 100 l of 1% sodium dodecyl sulfate (SDS) to each well.

7.Read the plates using the ELISA plate reader, set at a reading wavelength of 405 nm.

3.6.2 ELISA assay for detection of cytokines TC "3.6.2 ELISA assay for detection of cytokines" \l 2

Rather than using antigen as the capturing agent, the cytokine ELISA depends on using a cytokine-specific antibody which does not recognize the same cytokine epitope as the biotinylated detection antibody. Thus, monoclonal antibody (MAb) pairs are required and are available routinely from many commercial sources. Alternately, you can use a monoclonal in conjunction with a polyclonal anti-cytokine antisera. However, care must be taken to ensure than the antibodies/antisera employed will only recognize the cytokine of interest and that they will not inappropriately bind to immunoglobulin or other assay reagents and thereby give "false-positive" results.

Materials

Immulon-4 ELISA plates

pipettes/pipettors

experimental samples for cytokine assay

Reagents

cytokine capture and biotinylated detection antibody pairsELISA carbonate coating bufferPBST (PBS with 0.05% Tween 20)

DMEM-10% FCS

SA-horse radish peroxidase (SA-HRP)

2-2'-azino-di[3-ethyl-benzthiazoline sulfonate (6)] with H2O2 (ABTS;

1 component substrate f