8th SEMINAR
SEPARATION 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
Negative separation
Labeled the unwanted
cells (depletion)
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.
The cells become affected only by
the separation environment, hence
this is the preferred strategy in
functional examinations.
FICOLL-PAQUE DENSITY BASED
CELL SEPARATION
peripheral blood
(or buffy coat)
pipettig the „ring”
containing the
mononuclear cells to a new
tube to get rid of Ficoll
centrifugation
plasma
mononuclear
cells
(PBMC)
ficoll
pipetting cells on
ficoll
Neutrophil
granulocytes
Red blood cells
separated
cells
(from Google pictures)
(Nature Protocols http://www.nature.com/nprot/journal/v3/n6/images/nprot.2008.69-F1.jpg)
SEPARATION METHODS BASED ON THE
IMMUNOLOGICAL PROPERTIES OF THE CELLS
Magnetic-Activated Cell Sorting (MACS)
antigen specific antibody
paramagnetic
bead
MAGNET
MAGNET
column
depleting or
selecting
unlabeled cells
(negative
separation)
SEPARATION METHODS BASED ON THE
IMMUNOLOGICAL PROPERTIES OF THE CELLS
Fluorescence-Activated Cell Sorting (FACS)
NKT cells
Example:
NKT cell separation (CD3/CD56)
blood sample
NK cells
lymphocytes
T cells
The fluid stream break up
into droplets by the
vibration of the flow cell.
breakoff point
vibration (nozzle orifice of the flow cell)
+
+
+
+
+
+
+
+
+
Laser
+
charged deflection +
plate
+
+
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
-
--- 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
MEASURING LYMPHOCYTE ACTIVITY
For detection of immunodeficiencies affecting T and/or B cell functions
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
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 T CELL ACTIVATORS
Phaseolus vulgaris
Phytohaemagglutinin (PHA)
Concanavalin A (ConA)
Anti-CD3, Anti-TCR antibodies
Canavalia ensiformis
Pokeweed mitogen (PWM)
Staphylococcus protein A superantigen (SpA)
Epstein Bar Virus (EBV) (transforming)
Anti-IgM antibody
Phytolacca americana
POLYCLONAL B CELL ACTIVATORS
Receptor crosslinking
(immediate)
phosphorylation steps
Antigen receptors (TCR, BCR),
cytokine receptors, etc.
-
Western blot
-
flow cytometry
fluorescent microscopy
-
qRT-PCR  mRNA
Western blot  protein
Cytokine synthesis
-
i.c. cytometry
Cytokine secretion
- ELISA
- ELISPOT
(seconds-minutes)
i.c. Ca2+ increase
Gene activation
Lymphocyte activation
The examination often requires
specific Ag-Ab reactions
Viability/apoptosis
Cell division
-
dies specific to
dead cells
-
3H-thymidine
CFSE
MTT
Fluo-3
or
Indo-1
An increase in cytoplasmic Ca2+
levels can be detected by
fluorescent indicator dyes
/Fluo-3 or Indo-1/
Fluorescence proportional
with Intracellular Ca2+ level
MEASUREMENT OF CA2+ SIGNAL BY FLOW CYTOMETRY
activation
of cells
time
basic signal
INTRACELLULAR CYTOKINE DETECTION
BY IMMUNOFLUORESCENCE
cytokine specific
antibodies with
fluorescent labeling
 the cell membrane should
be permeabilized
(detergent)
 but first the cells should
be fixed to avoid
decomposition (using e.g.
aldehyde fixation)
cytokines
 optionally the cells can
also be labeled by cell
type (CD marker) specific
antibodies
INVESTIGATION OF GENE ACTIVATION
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)
the more mRNA the sample
contains, the less time (cycles)
it will take to reach the threshold
ELISPOT
Enzyme 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).
ELISPOT
Enzyme Linked Immuno-Spot
- coating with antigen specific capture antibodies
- blocking
- administration of the cells (activation, incubation)
- washing
- administration of biotin conjugated
antigen-specific secondary antibody
- avidin-enzyme conjugate
- administration of the insoluble
chromogenic substrate (AEC 3-amino-9-ethylcarbazol)
A spot showing
the place of the
cytokine
producing cell
Upper view of a well on an ELISPOT
plate with the generated spots
ELISPOT
Enzyme 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
thymidine- measures the increasing DNA content by β
decomposition, and does not answer the numbers of cell division,
and the dividing cell number.
3H-labeled
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.
CFSE
TRACKING 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)