8th SEMINARSEPARATION AND MEASUREMENT OF THE ACTIVITY OF IMMUNECOMPETENT CELLS
CELL SEPARATION
Physical isolation of the cells of interest from a heterogeneous population
Differences in the physical, biological or immunological properties of the cells are utilized to separate the cells. (Differences in cell surface receptor expression is often available – there is a possibility to further investigate the separated living cells).
physical – density, size cell biological – adherence, phagocytosis, sensitivity to the
medium immunological – antigen differences (surface marker)
Consideration taken to: purity, recovery, yield and viability of the cells
TWO SEPARATION STRATEGIES
Positive separation
Labeling and separation of
the cells of interest e.g. labeling a cell surface molecule by a
fluorescent antibody.
The cells become affected both by the separation environment and the antibodies bound to the receptors. The purity of the separation is generally high.
Negative separation
Labeled the unwanted cells (depletion)
The cells become affected only by the separation environment, hence this is the preferred strategy in functional examinations.
peripheral blood (or buffy coat)
pipetting cells on ficoll
centrifugation
separated cells
plasma
ficoll
Red blood cells
mononuclear cells
(PBMC)
Neutrophilgranulocytes
pipettig the „ring” containing the mononuclear
cells to a new tube to get rid of Ficoll
FICOLL-PAQUE DENSITY BASED CELL SEPARATION
(Nature Protocols http://www.nature.com/nprot/journal/v3/n6/images/nprot.2008.69-F1.jpg)
(from Google pictures)
Magnetic-Activated Cell Sorting (MACS)
paramagneticbead
antigen specific antibody
SEPARATION METHODS BASED ON THE IMMUNOLOGICAL PROPERTIES OF THE CELLS
MAG
NETM
AGN
ET
column
depleting or selecting
unlabeled cells(negative
separation)
Fluorescence-Activated Cell Sorting (FACS)
SEPARATION METHODS BASED ON THE IMMUNOLOGICAL PROPERTIES OF THE CELLS
Example:NKT cell separation (CD3/CD56)
NK cells
NKT cells
lymphocytes
blood sample
T cells
The fluid stream break up into droplets by the
vibration of the flow cell.
breakoff point
Laser
+ ----
+++
+++ +
+
--
---
vibration (nozzle orifice of the flow cell)
If the wanted cell reaches the breakoff point, the stream become charged for the short time of drop formation, so the formed drop become charged
charged deflection plate
charged deflection plate
++++++++
+
collection tube collection tube
waste
MEASURING THE ACTIVITY OF IMMUNECOMPETENT CELLS
PHAGOCYTIC CELLS – PHAGOCYTOSIS ASSAY
• Using killed pathogens (bacteria: E. coli, S. aureus; yeast: S. cerevisiae) labeled with different fluorophores
• Phagocytosis can be detected by fluorescent microscopy or by flow cytometry
The activation of lymphocytes by a specific antigen is hardly detectable (low numbers of the antigen specific cells)
The activation of lymphocytes by a polyclonal activator can help investigate abnormal lymphocyte functions
MEASURING LYMPHOCYTE ACTIVITY
For detection of immunodeficiencies affecting T and/or B cell functions
POLYCLONAL ACTIVATION OF B AND T CELLS
Lectins (like concavalin A and PHA) act through crosslinking receptors
Intracellular signaling cascade activators (PMA – PKC activator, Ionomycin – increased intracellular Ca2+ levels)
Specific antibodies (anti-IgM, anti-CD3, anti-TCR)
POLYCLONAL B CELL ACTIVATORS
POLYCLONAL T CELL ACTIVATORS
Pokeweed mitogen (PWM)
Staphylococcus protein A superantigen (SpA)
Epstein Bar Virus (EBV) (transforming)
Anti-IgM antibody
Phytohaemagglutinin (PHA)
Concanavalin A (ConA)
Anti-CD3, Anti-TCR antibodies
Canavalia ensiform
isP
hytolacca ame
ricana P
haseolus vulgaris
Receptor crosslinking(immediate)
phosphorylation steps(seconds-minutes)
- Western blot
i.c. Ca2+ increase - flow cytometry- fluorescent microscopy
Gene activation - qRT-PCR mRNA- Western blot protein
Cytokine synthesis
Cytokine secretion
- i.c. cytometry
- ELISA- ELISPOT
Antigen receptors (TCR, BCR), cytokine receptors, etc.
Viability/apoptosis - dies specific to dead cells
Cell division - 3H-thymidine- CFSE- MTT
Lymphocyte activationThe examination often requires
specific Ag-Ab reactions
Flu
ore
scen
ce p
rop
ort
ion
al
wit
h I
ntr
acel
lula
r C
a2+ l
evel
time
basic signal
activation of cells
Fluo-3or
Indo-1
MEASUREMENT OF CA2+ SIGNAL BY FLOW CYTOMETRY
An increase in cytoplasmic Ca2+ levels can be detected by fluorescent indicator dyes
/Fluo-3 or Indo-1/
INTRACELLULAR CYTOKINE DETECTION BY IMMUNOFLUORESCENCE
cytokine specific antibodies with fluorescent labeling
cytokines
the cell membrane should be permeabilized (detergent)
but first the cells should be fixed to avoid decomposition (using e.g. aldehyde fixation)
optionally the cells can also be labeled by cell type (CD marker) specific antibodies
Activation of cells can be monitored by the detection of mRNA transcription of the activated genes
e.g. activation of cytokine genes
QUANTITATIVE (REAL-TIME) PCR (qPCR/qRT-PCR)
cells RNA isolation RNA reverse transcription (RT-PCR) cDNA cDNA polymerase chain reaction (PCR)
determination of quantity
(investigation of gene activation on protein level WB)
INVESTIGATION OF GENE ACTIVATION
the more mRNA the sample contains, the less time (cycles) it will take to reach the threshold
ELISPOTEnzyme Linked Immuno-Spot
Similar principles as in ELISA
Determination of the number of cells that produce Ig, cytokines, chemokines, granzymes and other soluble effector molecules
Sensitive. Allows the determination of 1 activated cell among 300,000 others. (Can reveal activated effector cells not only after polyclonal but after antigen specific activation).
- coating with antigen specific capture antibodies
- blocking
- administration of the cells
- administration of biotin conjugated antigen-specific secondary antibody
- avidin-enzyme conjugate
- administration of the insoluble chromogenic substrate (AEC 3-amino-9-ethylcarbazol)
(activation, incubation)
- washing
A spot showing the place of the
cytokine producing cell
Upper view of a well on an ELISPOT plate with the generated spots
ELISPOTEnzyme Linked Immuno-Spot
ELISPOTEnzyme Linked Immuno-Spot
Spot number and size determination is valuated slowly and manually by microscopy or using “ELISPOT plate reader” which is fast and standardizable
VIABILITY ASSAYS
MTT (Dimethyl thiazolyl diphenyl tetrazolium salt)Colorimetric test for measuring viability (apoptotic cells). NADPH-dependent cellular oxyreductase enzymes that reduce MTT dye to an insoluble purple color (formazan).
PI (propidium iodide)A fluorescent molecule intercalating with nucleic acids for measuring cell viability by flow cytometry. It is impermeable to viable cells.
7-AAD (7-aminoactinomycin)A fluorescent chemical intercalating with dsDNA. Won’t pass intact cells so is used for cell viability by flow cytometry.
PROLIFERATION ASSAYS
3H-labeled thymidine- measures the increasing DNA content by β decomposition, and does not answer the numbers of cell division, and the dividing cell number.
Bromodeoxyuridin (BrdU) A Thymidine-analogue can be administered to experimental animals, or cell cultures, and the proliferating cells can be detected by labelling with BrdU specific antibody (microscopy, FACS).
CFSE (Carboxyfluorescein succinimidyl ester)A fluorescent dye easily penetrating cells binding intracellular amine structures for long periods. Studies of cell divisions, prolifearation, migration and positioning.
CFSETRACKING THE CELL DIVISIONS
„Cell tracer” dye enters the cell, and becomes trapped there
The apolar CFSE can bind covalently to the cellular proteins
Progressively halved within daughter cells
Used in vitro and in vivo to monitor lymphocyte proliferation
CFSE-labeled cells that were not treated with polyclonal activator
(control)