In Vivo CFSE 01

UNIT 4.9Use of the Intracellular Fluorescent DyeCFSE to Monitor Lymphocyte Migrationand Proliferation

The stable incorporation of the intracellular fluorescent dye 5-(and -6)-carboxyfluo-rescein diacetate succinimidyl ester (CFSE) into lymphocytes (Basic Protocol) is apowerful tool to monitor lymphocyte migration in vivo and to quantitatively analyze celldivision both in vivo and in vitro (Support Protocol). The stability of CFSE-labelingallows monitoring of lymphocytes over a period of months in vivo. Cell division resultsin sequential halving of fluorescence, and up to 8 divisions can be monitored before thefluorescence is decreased to the background fluorescence of unstained cells. The relation-ship between cell division and cell function is readily measured at the time of analysis byusing a cell function marker (cell surface or intracellular protein) labeled with an alternatefluorochrome. Similarly, T and B lymphocyte subsets and NK cells can be individuallyanalyzed for cell division in a complex population by using appropriate cell surfacemarkers. CFSE remains associated with apoptotic cells for several days, and these can beanalyzed together with live cells by appropriate electronic gating (by size and granularity)on the flow cytometer. Since halving of fluorescence occurs in daughter cells, bycalculating the proportion of cells in each division peak and dividing by the expectedprogeny at those divisions (2, 4, 8, 16, etc.), the number of cells that have entered divisioncan be calculated. This gives a precursor frequency estimate of responding cells in thecultures.

BASICPROTOCOL

CFSE-LABELING OF LYMPHOCYTES

This basic protocol describes methods for labeling high or low numbers of lymphocyteswith CFSE. Steps are provided to use CFSE-labeled cells in cell transfer studies or ascells to be cultured in vitro. If analysis of cell migration is a goal of the experiment, specificguidelines for positioning of CFSE-labeled lymphocytes in lymphoid organs or othertissues are provided in this protocol. A Support Protocol for flow cytometric analysis ofCFSE-labeled cells follows.

Materials

Experimental animals or human peripheral blood or cultured lymphocytesPhosphate-buffered saline( PBS; APPENDIX 2), pH 7.4Hanks’ balanced salt solution (HBSS), pH 7.4 (APPENDIX 2)PBS (APPENDIX 2) containing 5% (v/v) heat-inactivated FBS5 mM CFSE stock solution (see recipe)Antigens and mitogens of interest0.5 mM disodium EDTA in PBS (APPENDIX 2)Fluorescence microscope with filters for fluorescein

Additional reagents and equipment for removal of mouse lymphoid organs (UNIT

1.9), preparation of mononuclear cell suspensions (UNIT 3.1), and isolation ofperipheral blood mononuclear cells (UNIT 7.1), immunohistochemistry (UNIT 21.4),and culturing mouse (UNITS 3.10 & 3.12), or human (UNITS 7.10 & 7.11) lymphocytes

Label lymphocytes with CFSE1a. For high cell numbers: Prepare lymphocytes using the techniques described in UNIT

1.9 (for mice; removal of lymphoid organs), UNIT 3.1 (preparation of cell suspensions),and UNIT 7.1 (preparation of PBMC), at a concentration of 50 × 106 cells/ml in either

Supplement 49

Contributed by Christopher R. Parish and Hilary S. WarrenCurrent Protocols in Immunology (2001) 4.9.1-4.9.10Copyright © 2001 by John Wiley & Sons, Inc.

4.9.1

In Vivo Assays forLymphocyteFunction

PBS (without serum) for human PBMC or HBSS (without serum) for mouselymphocytes.

1b. For low cell numbers: Resuspend freshly isolated lymphocytes in PBS containing5% FBS at concentrations from 0.5 × 106 cells/ml to 10 × 106 cells/ml.

At low cell concentrations it is absolutely essential that there be protein present to bufferthe toxic effect of CFSE.

Cultured lymphocytes that are quiescent at the end of primary culture are labeled directlyin their culture medium (containing 10% FBS) after equilibrating to room temperature.

2. Dilute the stock 5 mM CFSE solution 1/100 in PBS (to give a 50 µM solution). Add110 µl of this solution per ml of cells (to give a final concentration of 5 µM), and mixrapidly. After 5 min at room temperature add 10 vol of PBS containing 5% FBS,centrifuge the cells 5 min at 300× g, 20°C, and remove the supernatant. Wash threetimes, each time by resuspending in 10 vol PBS containing 5% FBS, centrifuging 5min at 300 × g, 20°C, and removing the supernatant.

Labeling with CFSE occurs rapidly, and it is essential that CFSE be dispersed as evenlyand quickly as possible so that cells are uniformly labeled. One strategy to achieve this isto add the cell suspension into the bottom of a 10-ml plastic tube, then while holding thetube almost horizontally, add the CFSE solution to a non-wetted portion of the plastic atthe top of the tube. The tube is then capped while still in the near horizontal position, andthen rapidly inverted several times to mix the lymphocytes and CFSE solution. An alternatestrategy is to predilute the CFSE to 10 �M and add an equal volume to the cell suspensionwhile vortexing. If this strategy is used for high cell numbers, prepare the lymphocytes at100 × 106 cells/ml instead of 50 × 106 cells/ml.

When labeling cultured lymphocytes it is best to add CFSE directly into the existing culturemedium without prior centrifugation. When cultured cells are centrifuged they form smallaggregates such that individual cells are not exposed equally to CFSE. After labelingcultured lymphocytes with CFSE, the cells are washed in PBS and then incubated for 5 minin 0.5 mM EDTA in PBS to dissociate any aggregates, and washed once more in PBS beforeresuspending in culture medium for restimulation in culture.

CFSE staining of lymphocytes cannot be measured directly after labeling because of theextremely high fluorescence. The majority of CFSE initially taken up by the cells is notstably incorporated and is lost within the first few days.

Perform in vivo transfer of CFSE-labeled lymphocytes3. Resuspend CFSE-labeled lymphocytes in tissue culture medium lacking added

protein (no serum) and inject intravenously (i.v.) into recipient animals. In the caseof mice, inject into the lateral tail vein, with from 1 × 106 to 40 × 106 cells beinginjected into each recipient mouse in a volume of 0.1 to 0.2 ml.

There is a linear increase in the number of CFSE-labeled cells entering mouse lymphoidorgans with the transfer of up to 50 × 106 cells, but when greater numbers of cells aretransferred the system appears to become saturated.

If in vivo migration of lymphocytes is being investigated under conditions of minimal celldivision, it is possible to independently track two different lymphocyte populations in thesame animal by labeling the cells to different fluorescence intensities with CFSE (Lyons,1999). One population is labeled with 5 �M CFSE (see step 1a or b above) and the otherpopulation with one-quarter (1.25 �M) or one-sixteenth (0.312 �M) the normal CFSE-la-beling concentration.

If one plans to examine the CFSE-labeled cells less than 24 hr after they have beentransferred into recipient animals, in order to avoid off-scale fluorescence intensities onthe flow cytometer, the lymphocytes should be labeled with one-quarter (1.25 �M) orone-eighth (0.625 �M) the normal CFSE-labeling concentration (see step 1 or 2 above).

Supplement 49 Current Protocols in Immunology

4.9.2

Use of CFSE toMonitor

LymphocyteMigration and

Proliferation

4. Detect the positioning of CFSE-labeled lymphocytes within lymphoid organs andother tissues by fluorescence microscopy with filter settings for fluorescein, or byimmunohistochemistry (UNIT 21.4) using fluorescein-specific antibodies (Garton andSchoenwolf, 1996; Graziano et al., 1998). For fluorescence microscopy detection ofCFSE-labeled cells, remove organs from animals, cut ∼3-mm sections of the organswith a razor blade, and place the sections on microscope slides for examination.

CFSE fluorescence is rapidly quenched when tissue sections are viewed by fluorescencemicroscopy and is totally quenched when the sections are treated with conventionalhistological stains. For short-term positioning studies of relatively low resolution, theDNA-intercalating dye H33342 is recommended as a highly fluorescent, quenching-resis-tant dye for labeling lymphocytes (Parish, 1999).

If high-resolution positioning studies are required, immunohistochemical detection ofCFSE-labeled cells in tissue sections is recommended.

Culture lymphocytes5. Resuspend CFSE-labeled lymphocytes in culture medium and stimulate in vitro with

antigens or mitogens of interest.

Procedures for culturing mouse and human lymphocytes are detailed in UNITS 3.10 & 3.12,and UNITS 7.10 & 7.11, respectively.

Harvest cells6a. For in vivo harvesting: Collect lymphoid organs, and make single-cell suspensions

(UNITS 1.9 & 3.1).

6b. For in vitro harvesting: Harvest cells from culture, wash once in 3 ml PBS, resuspendin 2 ml 0.5 mM EDTA/PBS, and incubate for 5 min at 37°C to dissociate aggregates.Centrifuge cells at 5 min at 300 × g, 20°C, resuspend in PBS containing 5% FBS,and transfer to tubes suitable for use with the flow cytometer. If the cells are to becounted manually with a hemacytometer (APPENDIX 3A), retain a small sample on icein a separate tube or V-well plate.

Cells for analysis on the flow cytometer are not fixed with paraformaldehyde, but are kepton ice until analyzed.

For multiple cultures, it is convenient to use 96-well V-well plates. For cultures harvestedfrom 96-well flat well plates, an aliquot of supernatant from each of the wells is firstdiscarded so that the triplicate cultures can be combined into 1 well of the 96-V-well plate.Subsequent washing and treatment with EDTA then occurs in 150-�l volumes in these wells.The plates are centrifuged for 2 min at 300 × g in a plate holder. The supernatant is removedusing a microtiter pipettor by following the meniscus down while aspirating until the tipreaches the intersection of the vertical wall and the top of the V-base of the well. The pelletis mixed by agitating the plate over a vortex mixer prior to adding the next wash solutionwith a multipipettor.

SUPPORTPROTOCOL

ANALYSIS OF CFSE-LABELED CELLS BY FLOW CYTOMETRY

This protocol summarizes the steps used to analyze CFSE-labeled cells in order toquantitate cell division. The proportion of cells in the individual CFSE peaks can bedetermined manually or by applying software that deconvolutes the peaks. Guidelines foreither approach are provided in this protocol. The Critical Parameters section of this unitdescribes important parameters related to the analysis of CFSE-labeled cells that havebeen transferred in vivo or cultured in vitro following labeling. Additional informationabout flow cytometric analysis of cells can be found in Chapter 5. In this protocol, thechoice of which other labeling procedures are to be used (e.g., cell surface staining,

Current Protocols in Immunology Supplement 49

4.9.3


intracellular staining for cytokines, identification of apoptotic cells) depends on the goalsof the experiment at hand.

Materials

CFSE-labeled cells (see Basic Protocol)Analytical flow cytometer capable of 3-color fluorescenceCell sorting flow cytometer (for some applications)

Additional reagents and equipment for cell surface staining of lymphocytes (UNIT

5.3), intracellular cytokine staining (UNIT 6.24), analytical flow cytometry (UNIT

5.4), flow cytometric analysis of apoptosis (UNIT 3.17), and cell sorting flowcytometry (UNIT 5.1).

1. Perform flow cytometric staining steps suited to the experiment being performed(e.g., cell surface staining, UNIT 5.3; intracellular cytokine staining, UNIT 6.24; propidiumiodide staining for measurement of apoptosis, UNIT 3.17). Perform flow cytometricanalysis (UNIT 5.4).

CFSE is a fluorescein-based dye. Labeling lymphocytes for cell surface staining orintracellularly for cytokines requires the use of an alternative fluorochrome such as PE orCy5 (see Table 6.21.2).

For software-based calculations2a. Deconvolute CFSE peaks using appropriate software.

Several software programs, such as Profit for Macintosh (Quantumsoft), Peakfit for PC(SPSS Sciences), ModFit (Verity Software House; see Internet Resources), and CFSEModeler (Science Speak; see Internet Resources) are available for deconvoluting CFSEpeaks.

For manual calculations2b. Record the geometric mean fluorescence of the control CFSE stained cells and the

unstained cells of both the control and stimulated cultures.

For example, one might have a geometric mean fluorescence of 600 for CFSE staining ofcontrol unstimulated cells compared to an autofluorescence control of 2 for stimulatedunstained cells.

Note that the autofluorescence value of the unstained cells is greater for the dividingcompared to the control nondividing cells.

3b. Subtract the geometric mean fluorescence of the appropriate control from that of theCFSE control cells (for example, 600 − 2 = 598).

4b. Convert this value to its base 10 logarithm (in the above case, 2.776).

5b. Determine the geometric mean fluorescence of daughter populations. As these occurat 1/2, 1/4, etc. of the undivided peak, subtract 0.3 log10 units.

Thus, continuing with the above example, the successive peaks are at 2.476 (division 1),2.176 (division 2), 1.876 (division 3), 1.576 (division 4), 1.276 (division 5), 0.976 (division6), and 0.676 (division 7).

6b. Determine the boundaries of the peaks.

The boundaries are midway between successive peaks and are therefore 0.15 log10 unitseach side of the peak. So the lower (left) boundary for each peak is as follows: 2.626(undivided control cells), 2.326 (division 1), 2.026 (division 2), 1.726 (division 3), 1.426(division 4), 1.126 (division 5), 0.826 (division 6), 0.526 (division 7). Now determine theantilog of these figures: 423, 212, 106, 53.2, 26.7, 13.4, 6.7, 3.4. To these values are addedthe geometric mean autofluorescence (2 in this example) to give 425, 214, 108, 55.2, 28.7,15.4, 8.7, 5.4.


4.9.4



Proliferation

7b. Apply these markers using a flow cytometry program (such as Cell Quest, BectonDickinson) and determine the % of cells in each peak.

8b. Determine the actual number of cells in each peak from the manual counts or countingbead analysis.

9b. Divide the cell numbers in each peak by the expected progeny for those divisions.Thus divide by 2 for 1 division, divide by 4 for 2 divisions, divide by 8 for 3 divisions,etc.

The total of these numbers gives the number of progenitor cells and can be compared withthe number of cells in control cultures.

In some cases applying the above calculations gives markers that are not aligned exactlyover the peaks. When the calculations are redone using division 1 as a reference peak, themarkers then align correctly. It appears that during the transition from undivided to thefirst division there is some loss of incorporated CFSE. This appears to occur with somecell types more than others. The example given in Figure 4.9.1 for human peripheral bloodlymphocytes responding to phytohemagglutinin is a case in point. You will note that thegeometric mean for the undivided cells is 763 which is more than twice that of the peak indivision 1. The remainder of the peaks are close to the expected halving with each division.

This quantitative analysis of cell division giving the number of progenitor cells providesuseful information. First, the lymphocyte population may be stimulated to divide and to

Figure 4.9.1 CFSE profiles for PBMC labeled at 1 × 106/ml in PBS containing 5% FBS with 5 µMCFSE for 5 min and cultured with 5 µM phytohemagglutinin for 4 days. The unfilled peak on the rightat 7.6 × 102 fluorescence units corresponds to the control unstimulated CFSE-labeled cells. Theunfilled peak on the left at 2 × 100 fluorescence units corresponds to the autofluorescence ofunlabeled cells. The calculated marker positions are shown: M1 (undivided), M2 (1 division), M3 (2divisions), M4 (3 divisions), M5 (4 divisions), M6 (5 divisions), and M7 (6 divisions).


4.9.5


undergo activation-induced cell death. Dead cells retain the CFSE dye and thus the divisionnumber in which death of a cell occurred can be readily determined. Dead cells can beidentified by forward and side scatter or by propidium iodide uptake. Comparison of thenumber of progenitor cells in the stimulated and control cultures will show if significantcell death has occurred. Second, the percentage of progenitor cells in divisions 1 through7 compared to divisions 0 through 7 is an estimate of the precursor frequency, i.e., theproportion of the lymphocyte population with reactivity to a particular antigen or mitogen.In the experiment shown in Figure 4.9.1, 29.63% of cells have entered division. Thelimitation using this technique for the determination of precursor frequencies is the numberof divisions that occur before the fluorescence of the dividing cells merges with theautofluorescence of unstimulated cells.

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, seeAPPENDIX 2A; for suppliers, see APPENDIX 5.

CFSE solutionDissolve 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE; Mo-lecular Probes) in dimethylsulfoxide at a final concentration of 5 mM. Store insuitably sized aliquots at −20°C.

The stock solution can be refrozen provided this is done as soon as possible after thawing.Prepare the substock of diluted CFSE just before use. Do not refreeze the substock.

COMMENTARY

Background InformationUnlike most other cells in vertebrates, the

cells of the immune system have the remarkableability to continually migrate throughout thebody and position themselves in specific loca-tions within tissues, particularly within lym-phoid organs. In addition, following contactwith antigen, there is a rapid clonal expansionof antigen-specific T and B lymphocytes, withthe migration and positioning behavior of theproliferating lymphocytes often being very dif-ferent from their precursors.

In order to fully understand a functioningimmune system, techniques are required thatcan simultaneously follow lymphocyte prolif-eration, lymphocyte migration into differentlymphoid organs, and the positioning patternof the lymphocytes within lymphoid organs.Early studies employed radioactive markers tomeasure lymphocyte proliferation and followlymphocyte migration. [3H]thymidine has beenwidely used to follow lymphocyte prolifera-tion, both in vitro (UNIT 7.10) and in vivo. How-ever, this approach suffers from the disadvan-tage that it is difficult to assess the subpopula-tion of lymphocytes that is proliferating,tedious autoradiography procedures being re-quired to enumerate the proliferating cells. Fur-thermore, the procedure only measures thosecells that are in S phase at the time of[3H]thymidine addition. Bromodeoxyuridine

(UNIT 4.7) is an excellent reagent for measuringlymphocyte turnover in vivo but yields limitedmigration and positioning information. The γ-emitting isotope 51Cr has been used for manyyears to follow the distribution pattern of in-jected lymphocytes in vivo but has a number oftechnical limitations (Ford, 1978), and yieldsno information about the phenotype, prolifera-tion status and positioning pattern of the in-jected cells.

During the last 20 years, at least 14 differentfluorescent dyes have been used to monitorlymphocyte migration (Parish, 1999), with thefluorescein-based dye CFSE emerging as themost versatile fluorescent dye. CFSE is a mem-brane-permeant dye that can very stably labelcells by covalently coupling to intracellularmolecules. CFSE was originally developed asa long term tracking dye for lymphocyte migra-tion studies (Weston and Parish, 1990). It soonbecame apparent that a major advantage ofCFSE is that it can be used to monitor lympho-cyte proliferation, due to the progressive halv-ing of CFSE fluorescence in cells following celldivision (Lyons and Parish, 1994). Thus CFSEis an extremely valuable reagent for immu-nological studies, as lymphocytes labeled withCFSE can be simultaneously monitored fortheir proliferation status (either in vitro or invivo) and their migration and positioning be-havior in vivo.


4.9.6



Proliferation

There is some confusion in the literatureregarding the nomenclature for CFSE. Thecompound used to label cells is carboxyfluo-rescein diacetate succinimidyl ester, and thus amore appropriate acronym would be CFDA-SErather than CFSE. Unfortunately, when theauthors first obtained the dye from MolecularProbes in the late 1980s, it was listed in thecatalog as CFSE and this acronym was used inthe first publications from the authors’ labora-tory reporting the use of the dye. In more recentMolecular Probes catalogs, the dye is listed asCFDA-SE, but in order to be consistent andavoid confusion, the authors have continued touse the CFSE acronym in all of their publica-tions.

To fully appreciate the use of CFSE as a dyefor studying lymphocyte proliferation and mi-gration, it is important to understand the mo-lecular basis of cell labeling by CFSE. Figure4.9.2 depicts the various stages in the labelingof cells with CFSE. Carboxyfluoresceindiacetate succinimidyl ester (CFDA-SE, seeFig. 4.9.2), because of two acetate side chains,is nonfluorescent and highly membrane per-meant. Thus, this compound is rapidly taken upby cells, although due to its lipophilic nature

CFDA-SE also freely exits from cells. Onceinside cells, intracellular esterases remove theacetate groups. The resultant carboxyfluo-rescein succinimidyl ester (CFSE, see Fig.4.9.2) is highly fluorescent, and, due to itsreduced lipophilicity, it exits from cells at amuch slower rate. This slower exit rate alsoprolongs the time available for CFSE to cova-lently couple to intracellular molecules. Thesuccinimidyl side chain of CFSE is highly re-active with amino groups, resulting in carboxy-fluorescein (CF) being coupled, via a very sta-ble amide bond, to the amino groups of intra-cellular molecules. In some cases, CF iscoupled to molecules to form conjugates(CFR1) that are rapidly degraded or still exitthrough the plasma membrane, whereas inother cases the CF conjugates (CFR2) are stableand unable to exit from the cell (see Fig. 4.9.2).It is these long-lived conjugates that allow sta-ble labeling of cells with CFSE to be achieved.

During the last few years many laboratorieshave used CFSE to track lymphocyte cell divi-sion both in vitro and in vivo (reviewed inParish, 1999). An important feature of thesestudies is that many biological processes inlymphocytes, such as T cell cytokine produc-

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Figure 4.9.2 A schematic representation of the various stages in the labeling of cells withcarboxyfluorescein diacetate succinimidyl ester. For details see text. The size of arrows in the figureis proportional to the rate of diffusion of the different molecules through the cell membrane. Thecarbonyl group located between positions 5 and 6 on the benzene ring is to indicate that the dyeis a mixture of 5- and 6-carbonyl structural isomers. Figure reproduced from Parish (1999) with kindpermission of the publishers (Blackwell Science Asia).


4.9.7


tion, B cell Ig isotype switching, T cell apop-tosis, and cell surface molecule expression havebeen shown to be very division dependent.Recently a particular powerful application ofCFSE-labeling has been to monitor T lympho-cyte proliferation in response to a range ofcostimulatory signals (Gett and Hodgkin,2000). Based on their experimental data, Gettand Hodgkin derived an elegant mathematicalmodel for the assessment of the effect of differ-ent signals on T cell clonal expansion. The threekey variables that emerged as determining themagnitude of the T cell proliferative responsewere the time to first division, the subsequentrate of division, and the rate of apoptosis, withdifferent cytokines and costimulatory mole-cules having unique effects on these three pa-rameters. Although the majority of studies haveused CFSE to follow lymphocyte division, theproliferation of cell types as diverse as fi-broblasts and bacteria has been measured usingCFSE (Khil et al., 1997; Ueckert et al., 1997).CFSE-labeling has also been recently reportedfor tumor cell tracking (von Horsten et al.,2000), nuclei labeling (Hasbold and Hodgkin,2000), and hematopoietic stem cell prolifera-tion (Oostendorp et al., 2000).

Critical ParametersCFSE is toxic to cells at high concentrations,

and it is therefore essential to determine theoptimum labeling conditions that give goodfluorescence and preserve normal function. Forhuman peripheral blood lymphocytes at con-centrations below 10 × 106/ml, reliable labelingis best achieved with the cells resuspended inPBS containing 5% FBS and using CFSE at afinal concentration of 5 µM. These conditionsgive a fluorescence intensity between the sec-ond and third decade when measured at 4 days.Labeling exceeding the third decade gives im-paired functional responses. This is illustratedin Figure 4.9.3 showing the relationship, after4 days culture, between the amount of CFSE incontrol unstimulated human PBMC and theresponse of the CFSE-labeled cells to stimula-tion with 5 µM phytohemagglutinin. This datais a compilation of that obtained in an experi-ment comparing labeling at different CFSEconcentrations (1 µM to 7.5 µM) and lympho-cyte concentrations (0.5 × 106 to 50 × 106/ml)and in the presence or absence of added protein.For any given CFSE concentration, the pres-ence of protein reduces the amount of CFSEincorporated. Labeling is proportional to CFSE

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4.9.8



Proliferation

concentration and is essentially complete by 5min.

Uniform labeling with CFSE is essential toobtain clearly defined peaks following divi-sion. That is, the coefficient of variation (CV)of the CFSE-labeled control unstimulated lym-phocytes must be small. This is in part deter-mined by the uniformity in cell volume of thepopulation, and in part by the technique formixing CFSE with the cells. For some applica-tions it is useful to sort the cells for uniformstaining following CFSE-labeling (Nordon etal., 1999) prior to culture. Cultured lympho-cytes can also be labeled with CFSE, but it isessential to label them when they have revertedto small cells and are quiescent. CFSE-labelingof cultured human NK cells has allowed ananalysis of cell surface receptors that stimulatedivision in secondary culture (Warren, 1999;Warren and Kinnear, 1999).

Flow cytometryAnalysis of in vivo–transferred cells. When

examining CFSE-labeled lymphocytes in cellsuspensions from in vivo experiments, ideally0.5–1 × 106 events are collected, with care beingtaken not to gate out the highly fluorescentCFSE-labeled cells during data acquisition. Analternative approach, which reduces data-stor-age requirements, is to initially collect 25,000events in order to calculate the percentage ofdonor CFSE+ cells in the sample. One shouldthen collect 5000 CFSE+ events separately, us-ing electronic gating, to allow an assessment ofthe division status and phenotype of CFSE-la-beled cells.

With in vivo experiments the number ofevents that need to be collected for analysisdepends on three key factors: the number ofCFSE-labeled cells that have been injected, thetime since cell transfer, and the organ beingexamined. Usually, the highest proportion ofCFSE-labeled lymphocytes is detected in theperipheral blood and spleen, with a relativelylow percentage of cells being CFSE+ in thePeyer’s patches and peripheral lymph nodes.These considerations will influence the numberof events that need to be collected to accuratelydetermine the percentage of cells that areCFSE+. Gating on CFSE+ cells and the collec-tion of 5000 events is a convenient means ofacquiring information about the CFSE+ lym-phocytes in cell suspensions, particularly whenthere is a low frequency of CFSE+ cells.

Analysis of in vitro–cultured cells. If re-quired, add a known number of fluorescentcounting beads to the sample prior to collection

on the flow cytometer to allow subsequentenumeration of cells in individual CFSE peaks.Suitable beads are CaliBRITE beads (Becton-Dickinson) and Flow-Count fluorospheres(Beckman Coulter). When analyzing the data,apply appropriate electronic gates to view in-dividually the live cells, apoptotic cells, andcounting beads. The ratio of bead number tocell number gives the actual cell concentrationin the sample.

For acquisition on the flow cytometer, thelogarithmic mode for the side scatter (granular-ity) parameter must be used with countingbeads. Collect all events so that the data setincludes beads, apoptotic cells and live cells.

Apply the appropriate compensation valuesif lymphocytes are stained with PE orpropidium iodide. Collect the samples at nomore than 700 events per sec. Collect a suffi-cient number of events so that the proportionof cells in each peak can be accurately deter-mined. For control cultures where cells havenot divided, it is adequate to collect 5,000 to10,000 events. For cultures where cells havedivided it is a good idea to collect as many aspossible, e.g., 25,000 events.

Comparative studies of CFSE-labeled lym-phocytes maintained in vitro or in vivo haveshown that the labeled cells have identical fluo-rescence-intensity characteristics; i.e, CFSE-labeled lymphocytes lose the CFSE label at thesame rate whether maintained in vitro or in vivo(Lyons, 1999).

Anticipated ResultsDuring the first few days following CFSE

labeling there is rapid decline in CFSE fluores-cence, presumably due to the loss of many CFconjugates from the cells. Despite this loss influorescence, there are sufficient amounts oflong-lived conjugates remaining to allow non-dividing CFSE-labeled lymphocytes to betracked in vivo for up to 6 months followinginjection. If lymphocyte proliferation is to bemonitored, up to eight divisions can be dis-cerned for at least a week after cell transfer orculture initiation. The slow decline in fluores-cence makes it increasingly difficult to measurelarge numbers of cell divisions in ensuingweeks.

Time ConsiderationsFor a moderate-size experiment, the isola-

tion of lymphocytes, labeling, washing andsetting up of lymphocyte cultures is accom-plished in ∼4 hr. Harvesting and washing cellsafter culture takes ∼1 hr, an additional 2 to 3 hr


4.9.9


would be required for cell surface or intracel-lular cytokine staining. Collecting cells on theflow cytometer and data analysis take ∼4 hr.

Literature CitedFord, W.L. 1978. The preparation and labeling of

lymphocytes. In Handbook of Experimental Im-munology, Vol. 3 (D.M. Weir, ed.) p. 23.1. Black-well Scientific, Oxford.

Garton, H.J. and Schoenwolf, G.C. 1996. Improvingthe efficacy of fluorescent labeling for histologi-cal tracking of cells in early mammalian andavian embryos. Anat. Rec. 244:112-117.

Gett, A.V. and Hodgkin, P.D. 2000. A cellular calcu-lus for signal integration by T cells. Nature Im-munol. 1:239-244.

Graziano, H.J., St-Pierre, Y., Beauchemin, C., Des-rosiers, M., and Potworowski, E.F. 1998. Thefate of thymocytes labeled in vivo with CFSE.Exp. Cell Res. 240:75-85.

Hasbold, J. and Hodgkin, P.D. 2000. Flow cytomet-ric cell division tracking using nuclei. Cytometry40:230-237.

Khil, L.Y., Kim, J.Y., Yoon, J.B., Kim, J.M., Keum,W.K., Kim, S.T., Yoon, Y., Yoon, M.Y., Moon,C.K., Lee, J.H., Ha, J., Kim, S.S., and Kang, I.1997. Insulin has a limited effect on the cell cycleprogression in 3T3 L1 fibroblasts. Mol. Cell7:742-748.

Lyons, A.B. 1999. Divided we stand: Tracking cellproliferation with carboxyfluorescein diacetatesuccinimidyl ester. Immunol. Cell Biol. 77:509-515.

Lyons, A.B. and Parish, C.R. 1994. Determinationof lymphocyte division by flow cytometry. J.Immunol. Methods 171:131-137.

Nordon, R.E., Nakamura, M., Ramirez, C., andOdell, R. 1999. Analysis of growth kinetics bydivision tracking. Immunol. Cell Biol. 77:523-529.

Oostendorp, R.A., Audet, J., and Eaves, C.J. 2000.High-resolution tracking of cell division sug-gests similar cell cycle kinetics of hematopoieticstem cells stimulated in vitro and in vivo. Blood95:855-862.

Parish, C.R. 1999. Fluorescent dyes for lymphocytemigration and proliferation studies. Immunol.Cell Biol. 77:499-508.

Ueckert, J.E., Nebe von-Caron, G., Bos, A.P., andter Steeg, P.F. 1997. Flow cytometric analysis ofLactobacillus plantarum to monitor lag times,cell division and injury. Lett. Appl. Microbiol.25:295-299.

von Horsten, S., Helfritz, A., Kuhlmann, S., Nave,H., Tschernig, T., Pabst, R., Ben-Eliyahu, S.,

Meyer, D., Schmidt, R.E., and Schmitz, C. 2000.Stereological quantification of carboxyfluo-rescein-labeled rat lung metastasis: A newmethod for the assessment of natural killer cellactivity and tumor adhesion in vivo and in situ.J. Immunol. Methods 239:25-34.

Warren, H.S. 1999. Using carboxyfluoresceindiacetate succinimidyl ester to monitor humanNK cell division: Analysis of the effect of acti-vating and inhibitory class I MHC receptors.Immunol. Cell Biol. 77:544-551.

Warren, H.S. and Kinnear, B.F. 1999. Quantitativeanalysis of the effect of CD16 ligation on humanNK cell proliferation. J. Immunol. 162:735-742.

Weston, S.A. and Parish, C.R. 1990. New fluores-cent dyes for lymphocyte migration studies:Analysis by flow cytometry and fluorescencemicroscopy. J. Immunol. Methods 133:87-97.

Key ReferencesGett and Hodgkin, 2000. See above.

This paper is an elegant demonstration of the powerof CFSE to monitor lymphocyte division using amathematical model for assessing the response oflymphocytes to different signals.

Lyons and Parish, 1994. See above.

This is the original paper describing the use ofCFSE to monitor lymphocyte division.

Parish, 1999. See above.

This paper reviews a range of fluorescent dyes fortheir use in lymphocyte migration and analysis ofcell division.

Internet Resourceshttp://www.vsh.com/

ModFit LT software is an excellent software for cellproliferation analysis and is available for Macin-tosh and PC.

http://www.sciencespeak.com

CFSE Modeler software has been designed specifi-cally for CFSE analysis, but is currently availableonly for Macintosh.

Contributed by Christopher R. ParishAustralian National UniversityCanberra, Australia

Hilary S. WarrenThe Canberra HospitalWoden, Australia


4.9.10



Proliferation

Documents

In Vivo CFSE 01