Applications of flow cytometry in basic immunology Generation and characterization of DC Assays for...
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Transcript of Applications of flow cytometry in basic immunology Generation and characterization of DC Assays for...
• Applications of flow cytometry in basic immunology
• Generation and characterization of DC
• Assays for T cell activation– Cell proliferation – Cell division – Cell activation marker
Laboratory demonstration - DC and T cell activation
– Basic parameters
– Applications• Cell phenotyping• Measurement of cell activation• Visualization of cell division
• Detection of intracellular molecues (e.g. cytokine)• Analysis of specific ligand binding• Analysis of intracellular calcium concentration• Analysis of DNA content • Cell sorting
Applications of Flow Cytometry in Basic Immunology
• FSC (forward scatter) - Size• SSC (side scatter) - Granularity• FL (flurochrome) – FL-1, FL-2, FL-3 etc.
Flow cytometry - the basic parameters
(1) FSC – the size
Forward (parallel) lights
(2) SSC – the granularity
Side (angled) lights
(3) Fluorescent-labeled markers
Laser light
BandFilters
mirr
ors
FL-1 (green)
FL-3 (ultra red)
FL-2 (red)
Fluorescent detectors
Fluorochromes & wave length
Cell types of the immune system
Human blood cells analyzed by their FSC and SSC properties
FSC (size)
SSC
(gra
nula
rity)
RBC lysedTotal blood
Cell phenotypes & surface markers
T B NK
Cluster of Differentiation (CD) markers
Immuno-staining of cell surface markers
Direct Indirect
Human blood leukocytes (back-gating analysis)C
D14
-FIT
C
CD45-PE FSC
SS
C
Human PBL phenotyping
Isotype-FITC
Isot
ype-
PESS
C
Anti-CD3-FITCAnti-CD3-FITCFSCA
nti-C
D19
-PE
Ant
i-CD
8-PE
Ant
i-CD
4-PE
Ant
i-CD
56/1
6-PE
Anti-CD3-FITC Anti-CD3-FITC
Markers of T cell differentiation
CD4 and CD8 expression on thymocytes, splenocytes and PBL
Thymocytes Splenocytes PBL
CD8-FITC
CD
4-C
yt5
SBT CT
CD25
CD
4
Regulatory T cells (triple stain)
CD45RCS
SC
Regulatory T cell:CD45RClo
CD4+
CD25+
(courtesy of Dr. Lina Tian)
CFSE: Carboxyfluorescein diacetate Succinimidyl Ester
Analysis of cell division by CFSE labeling
Cell incubation with CFSE(serum free, 37 0C, 10mins. & wash)
CFSE crosses cell membrane & attaches to cytoplasmic proteins
Removal of carboxyl groups of CFSE by intracellular esterase
Fluorescein Analyed by FC
Excitation wave length: 488nmEmitting wave length: 519nm
1
½
¼...
Visualizing B cell division
CFSE intensity
• Applications of flow cytometry in basic immunology
• Generation and characterization of DC
• Assays for T cell activation– Cell proliferation – Cell division – Cell activation marker
Laboratory demonstration - DC and T cell activation
Mitogen (Con A)-induced T cell proliferation(measured by level of DNA synthesis)
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
0 0.625 1.25 2.5 5 10 15
Con A Concentration ( g/ml)
Cell activation and proliferation analyzed by the FSC and SSC properties
FSC (size)
SSC
(g
ranu
larit
y)
Unstimulated Con A-stimulated
Dot plots
Density plots
T cell activation analyzedby CD25 (IL-2R) expression
CD25-PE
Cel
l num
ber
Unstimulated Con A-stimulated
SSC
FSC
Visualizing T cell activation
CFSE intensity
Cel
l num
ber
SS
C (g
ranu
larit
y)
FSC (size)
Unstimulated Con A-stimulatedCFSE-labeled unlabeled
Visualizing & Phenotyping T cell Division
CFSE intensity
Cel
l num
ber
CD25-PE
AB
A
B
Mitogen-induced T cell proliferation, division vs IL-2R (CD25) expression
CFSE intensity
CD
25
Unstimulated Con A-stimulated
DC:T cell interaction
(Huang YG, unpublished data)
Isot
ype
IsotypeM
HC
Cla
ss II
CD
80
CD86
CD40
- LPS + LPS
67.214.0
DC maturity & APC activityin Mixed Lymphocyte Reaction (MLR)
0
50000
100000
150000
200000
250000
1:1 1:5 1:10 1:20 1:30 1:50 1:100 1:200
Responder (SpC) Ratio
3H-
Thym
idin
e Up
take
(cpm
)
Mature DCImmature DC
Key points for discussions Requirements for naïve T cell activation?
Key changes during DC maturation?
Association of DC maturity with APC activity?
Induction and measurement of T cell activation – the basis - cell proliferation assay? - indicators of T cell activation? - T cell mitogen?
MLR - peptides presented to T cells in: - allogenic responses? - semi-allogenic responses? - syngenic responses?
Isot
ype
Isotype
MH
C C
lass
IIC
D80
CD86
CD40
GM-CSF GM-CSF+IL-4 GM-CSF + IL-4 GM-CSF + IL-4 (TW) (TW + LPS)
7.2 67.248.714.0