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Separation of the immune competent cells
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)
physical – density, size
cell biological – adherence, phagocytosis,
immunological – antigen differences
base strategies:
positive separation – labeling and separation of the cells of interest
negative separation – get rid of the labeled unwanted cells (depletion)
• Important parameters:
• purity
• recovery, yield
Ficoll-Paque: density based cell separation
peripherial blood
pipettig the „ring”
containing the
mononuclear cells to
a new tube
centrifugation
To dilute ficoll
plasma
mononuclear
cells
(PBMC)
ficoll
pipetting cells on ficoll,
or pipetting ficoll under
the cells
thrombocytes
granulocytes
Red blood cells
separated cells
Isolating or depleting adharent cells
Cheap, simple, but only for adharent cells.
Low purity and recovery.
Antibody ”panning”
coated antibodies
Complement mediated lysis
antibodies
complement
LYSIS
(Red blood cells could be lysated in mild hypotonic
ammonium-chloride buffer without any pretreatment)
Simple magnetic cell separation
Phagocyte cells can uptake small iron particles.
These cells could be separated with a strong magnet.
Magnetic immunoseparation (MACS)
antigene specific antibody
paramagnetic
bead
MACS
Magnetic cell separation (MACS)
separation of labeled cells
(positive separation)
CliniMACS – closed system
MAGNET
MAGNET
column
depleting or selecting
unlabeled cells
MicroBeads are very small, usually don’t interfere
with cellular functions.
CD8+ T cells
Flow cytometry
Most cells in the immune system can be found in free or loosely
adherent form. They can be labeled in cell suspension by
fluorescent antigen specific antibodies, and then they can be
examined cell by cell.
Cells flow in high velocity one by one through a light beam. The
light scatter and immunofluorescent properties of the cells are
collected and summarized in statistical manner.
The method provide qualitative and quantitative data – it can
detect the presence of different antigens in the cell, the
expression level of this antigen. Changes in the expression of
certain molecules can be followed after different treatment of
the specimen.
Example Chanel Layout for Laserbased Flow Cytometry
The emited fluorescent light can
be separeted to components by
special mirrors and filters
photodetectors
PMT 4
cell
suspension
in tube
flow cell
forward light scatter
detector
PMT 3
pl. PE
PMT 1
PMT 2
pl. FITC
side light scatter
detector
Laser
(PMT=photo-multiplayer tube)
Fluidics System
sheet fluid reservoir
Flow cell
Injector
+++
+++
+++
sheet
fluid
sample
6-10m/s flow rate
Fluorescent
light
Focused laser
beam
Fluorescence
Laser
FALS Sensor
FSC
Fluorescence detectors
(PMT3, PMT4 etc.)
autofluorescence - piridins and flavins
Becton Dickinson cytometers
from the ’80-ies and ’90-ies
benchtop flow cytometer
high speed sorter-flow cytometer
(FACS, FACS station)
30,000 cells/sec
Characterisation of immune cells using cell
surface markers
Cell types, differentiation stages can be identified using a
combination of cell surface markers.
Used in diagnostics:
- ratio of different cell types
- altered expression of cell surface markers
Examples:
- Inflammatory processes – increased neutrophil numbers
- HIV progression – decrease of CD4+ T cell count
CD4+ : CD8+ = 1.6
Normal CD4+ T cell count = 600 – 1400/l
AIDS = CD4+ T cell count <200/l
- increase of CD5+ B cells – typical for some B cell Leukemias
WAS: Wiscott-Aldrich Syndrome
Lack of CD43 expression
XLA: X-linked Agammaglobulinemia
Inhibited B cell development  lack of CD19
CD antigen
cell type
function
ligand
CD3
T cells
TCR signalling
-
CD4
helper T sejtek, (monocytes,
T cell coreceptor, (HIV
receptor)
MHC- II, HIV
pDC)
CD5
T cells, (B cell subset: B1)
adhesion, activation signals
CD72
CD8
cytotoxic T cells, (NK,  T cells)
T cell coreceptor
MHC I
CD14
monocytes, macrophages,
some granulocytes
LPS binding
LPS, LBP
CD19
B cells
part of CR2, B cell coreceptor
C3d, C3b
CD28
T cells
costimulatory signals to T
cells
(B7-1, B7-2)
CD80, CD86
CD34
hematopoietic progenitor cell
adhesion
CD62L
(L-selektin)
CD56
NK cell, (T and B cell subset)
homoadhesion (N-CAM
isoform)
APC: DC, B, monocyte,
macrophage
costimulatory signals
CD80, CD86
(B7-1, -2)
CD28, CD152
Different cell types - characteristic light dispersions
granulocytes
side light dispersion
(SSC)
(e.g. granulated)
monocytes
lymphocytes
forward light
dispersion (FSC)
(„size”)
The method for erythrocyte exemption affects
the distribution of remaining cell types
Lysis of erythrocytes
Ficoll-Paque density-based
separation
missing
granulocytes
”Gating” of different cell populations
granulocyte „gate”
monocyte „gate”
lymphocyte „gate”
Immunophenotyping
Example:
Measurement of CD4+ (helper) and CD8+ (cytotoxic) T cell
ratio (eg. monitoring AIDS progression)
(based on primari antibody-antigen reactions)
Labeling:
FITC labeled anti-CD4 antibody(α-CD4-FITC)
PE labeled anti-CD8 antibody (α-CD8-PE)
Th
NK
Tc
Lymphocytes in the periferial blood
B
detecting CD4-FITC
labeled (TH) cell
high velocity flow stream
(in cuvette or stream in air)
detector
signal
processing unit
CD8
PE
Screen
increasing light
intensity
a dot representing a
CD4+ CD8- cell
CD4
FITC
detecting the PE labeled cell
(CD8-PE)
CD8
PE
detector
signal
processing unit
increasing light
intensity
CD4
FITC
detecting the unlabeled cell
(eg.B cell) by autofluorescence
CD8
PE
detector
jelfeldolgozó
signal
processing
egység unit
CD4
FITC
CD8
PE
18%
44%
0%
quadrant
statistics
CD4 38%
FITC
Graphical representations 1.
dot-plot
contourplot
densityplot
Graphical representations 2.
Histogramm
FACS (Fluorescence Activated Cell Sorting)
any distinctive cell population can be gated, and
the gated cells could be separated
Different gating strategies can be combined:
• by light scattering
• by specific immunofluorescence
PMT 4
Sample
PMT 3
Flow
cell
PMT 2
PMT 1
Laser
Example:
•CD5+ B1 cell separation (CD19/CD5)
•NKT cell separation (CD3/CD56)
NKT cells
NK cells
lymphocytes
The fluid stream break up into dropplets by the
vibration of the flow cell.
breakoff point
vibration (nozzle orifice of the flow cell)
+
+
+
+
+
+
+
+
+
Laser
+
charged deflection +
plate
+
+
If the wanted cell reach the
breakoff point, the stream
become charged for the
short time of drop formation,
and the formed drop become
charged
+
+ +
+ +
- charged deflection
plate
-
--- collection tube
collection tube
waste