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Amina Kariminia Ph.D.
Oncology Lab, Child & Family Research Institute,Department of Pediatrics, Faculty of Medicine
University of British Columbia
ICIA 2014 Milad Tower, Tehran
Outlines• A glance to multi-color flow cytometry
• Tregs in whole blood sample
• Kinetic of NFkB phosphorylation in pre B-cell leukemia
• Monitoring immuno-suprressor drugs
• Multi-color flow cytometry for normal human B-cell sub-populations
• MIFlowCyt
• Introducing Cy-TOF: mass-cytometry
Types of controlsTypes of controls
• Instrument set up control– PMT voltage settings– Compensation
• Gating controls– Isotype controls– FMO controls
• Biological controls– Unstimulated samples– Healthy donors
Fluorescence Minus One is not always an ideal controlFluorescence Minus One is not always an ideal control
FMO FMO quadrant copy/pasted CD4- quadrant setting
CD3
Gal-Ser tetramer
No FRET with proper panel of conjugated Abs
No FRET with proper panel of conjugated Abs
Flow cytometry of rare populations Flow cytometry of rare populations
Minimal Residual Disease
GFP
PBL stem cells
BM stem cells
Tetramers: Ag specific T cells
Minimal Residual Disease
GFP
PBL stem cells
BM stem cells
Tetramers: Ag specific T cells
Rare population & Flow cytometryPractical Considerations
Rare population & Flow cytometryPractical Considerations
Number of events should be increased for statistical accuracy 200,000-1,000,000
Proper controls
Proper instrument setting
Number of events should be increased for statistical accuracy 200,000-1,000,000
Proper controls
Proper instrument setting
Intracellular stainingIntracellular staining
Cytokine
&
Phospho protein
Cytokine
&
Phospho protein
Intracellular stainingIntracellular staining• Cytokines
Type of stimulator Time point Type of protein transport inhibitor Level of production Background level Type of Fix/perm Color selection
• Cytokines
Type of stimulator Time point Type of protein transport inhibitor Level of production Background level Type of Fix/perm Color selection
Intracellular stainingIntracellular staining
• TH1/2/17
Frozen PBMCs from healthy donors PMA/I stimulation for 6 hr , CO2 incubator In presence of monensin 6 color staining BD fix/perm 500,000 cells acquired
• TH1/2/17
Frozen PBMCs from healthy donors PMA/I stimulation for 6 hr , CO2 incubator In presence of monensin 6 color staining BD fix/perm 500,000 cells acquired
CD3
CD4
IFN-g
IL-17
IFN-g
IL-4
Phospho-protein detectionPhospho-protein detection
• Phospho-protein
Type of stimulator Time point Level of production Background level Type of Fix/perm Color selection
• Phospho-protein
Type of stimulator Time point Level of production Background level Type of Fix/perm Color selection
B-cell Acute lymphoblastic leukemia cell linesB-cell Acute lymphoblastic leukemia cell lines
• Phospho-protein
TLR2 ligand Pam2CSK
Pre B-cell leukemia cell lines
Phosphorylation of NFkB & degradation of IkB
• Phospho-protein
TLR2 ligand Pam2CSK
Pre B-cell leukemia cell lines
Phosphorylation of NFkB & degradation of IkB
Time 0
Time 20Time 10
Time 5
380
0
5
10
15
20
25
30
0 0.01 0.1 1 10
TLR-2 agonist ug/ml
% of pNFkB+IKBa-
Pam2
Pam3
OP-1
0
5
10
15
20
25
30
35
40
45
0 0.01 0.1 1 10
TLR-2 agonist
% of pNFkB+IKBa-
Pam2
Pam3
TLR1
TLR-2
TLR-6
TLR1
TLR-2
TLR-6
TLR1
TLR-2
TLR-6
Dose response curve and phospho-protein
kinetic analysis
Dose response curve and phospho-protein
kinetic analysis
B-cell acute lymphoblastic leukemia: IL-7R expression
B-cell acute lymphoblastic leukemia: IL-7R expression
CD19+CD10+
0 102 103 104 105
<PE-A>
0
102
103
104
105
Sample %
Specimen_001_379-CD127.fcs 10.7
Specimen_001_1279-CD127.fcs 3.14
IL-7Ra
Ig Light chainHealthy control
B-ALL relapse
YB-1YB-1
Interruption of host immune system recognitionBy HLA-DO up-regulation/decreasing
Ag-presentation Prevention of apoptosis by Survivin
Blocking the effect of cytotoxic drugsThrough IL-7 signaling and multi-drug resistance -1 (MDR-1) up-regulation
YB-1 in acute lymphoblastic leukemia YB-1 in acute lymphoblastic leukemia
Novel IL-7 signaling pathway in B-cell ALL Phosphorylation of YB-1
Novel IL-7 signaling pathway in B-cell ALL Phosphorylation of YB-1
0 102 103 104 105
<PE-Cy7-A>
0
102
103
104
105 0.0141 0.243
94.65.09
0 102 103 104 105<PE-Cy7-A>
0
102
103
104
105 0 0.0237
94.85.17
CD19
pYB-1 No IL-7 IL-7 stimulated
Treg 6-color stainingCD3-PECY7, CD4-PerCPCY5.5, CD25-PE, CD127-FITC, CD45RA-V450, FOXP3-AF647
Treg 6-color stainingCD3-PECY7, CD4-PerCPCY5.5, CD25-PE, CD127-FITC, CD45RA-V450, FOXP3-AF647
Immunophenotyping on preserved whole blood sample usingCyto-Chex BCT tube
Immunophenotyping on preserved whole blood sample usingCyto-Chex BCT tube
0 102 103 104 105
<Qdot 800-A>: CD27
0
102
103
104
105
0 102 103 104 105
<APC-A>: CD10
0
102
103
104
105
51.5
11.8
6.98
0 102 103 104 105
<V500-A>: IgD
0
102
103
104
105
0 102 103 104 105
<Qdot 800-A>: CD27
0
102
103
104
105
B-cell ImmunophenotypingB-cell Immunophenotyping
0 102 103 104 105
PE-A
0
102
103
104
105
29.9
0 102 103 104 105
PE-A
0
102
103
104
105
0 102 103 104 105
APC-A
0
5
10
15
20
25
CD27-PE
IgD-FITC
CD19+ (Pacific Blue) gated cells
CD10-APC
IgD-FITC
CD27-PE
Bone marrow Peripheral Blood
Whole blood Treg phenotyping using eBioscience KitCD3-APCH7, CD4-AF700, CD25-PE, Helios-AF488, FOXP3-V450
Whole blood Treg phenotyping using eBioscience KitCD3-APCH7, CD4-AF700, CD25-PE, Helios-AF488, FOXP3-V450
0 102 103 104 105
<V450-A>: FOXP3
0
102
103
104
105
0 102 103 104 105
<V450-A>: FOXP3
0
102
103
104
105
Cyto-Chex tube EDTA tubeCD3+CD4+ gated T cells
0 102 103 104 105
<Alexa 647-A>
0
102
103
104
105
4.26
0 102 103 104 105
<Alexa 647-A>
0
102
103
104
105
1.82
FOXP3-AF647
CD25-PE
Treg phenotyping and drug monitoring of an unique IPEX caseCD3-AF488, CD4-PerCPCy5.5, CD25-PE, CD127-BV421, FOXP3-AF647
Treg phenotyping and drug monitoring of an unique IPEX caseCD3-AF488, CD4-PerCPCy5.5, CD25-PE, CD127-BV421, FOXP3-AF647
HD IPEX
0 102 103 104 105
<Pacific Blue-A>
0
102
103
104
105
0 102 103 104 105
<Pacific Blue-A>
0
102
103
104
105
CD25-PE
CD127-BV421
HD IPEX
Immuno-supressor Monitoringdetection of surface CD69 up-regulation
upon CD2 activation (IPEX patient)
Immuno-supressor Monitoringdetection of surface CD69 up-regulation
upon CD2 activation (IPEX patient)
CD3+CD4+ gated T cells
Un-stimulated
Corticosteroid Sirolimus
0 102 103 104 105
V450-A: FOXP3
0
102
103
104
105 7.04 1.07
17.374.5
0 102 103 104 105
V450-A: FOXP3
0
102
103
104
105 31.1 5.62
13.849.4
0 102 103 104 105
V450-A: FOXP3
0
102
103
104
105 11.1 2.31
10.576.1
Stimulated
The Minimum Information about Flow cytometry Experiment
(MIFlowCyt)
The Minimum Information about Flow cytometry Experiment
(MIFlowCyt)
Ref: Cytometry part A
73A:926-030
2008
Ref: Cytometry part A
73A:926-030
2008
MIFlowCytMIFlowCyt
Simplify cross-platform comparison and integration,
Simplify cross-platform comparison and integration,
MIFlowCytMIFlowCyt• Experimental overview:
– Purpose/goal hypothesis
– Experiment variables
– Conclusions
– Quality control
MIFlowCytMIFlowCyt
• Flow sample (specimen):
– Material
– Source/organism/location
– Treatment
– Reagent/analyte/detector/reporter
MIFlowCytData presentation
MIFlowCytData presentation
• Experimental and Sample Information:
– Experimental design
– Sample preparation reagents
– Fluorescent reagents• Vendor• Catalog number• Clone
MIFlowCytData presentation
MIFlowCytData presentation
• Data acquisition:
– Instrument configuration
– Instrument identification
– Fluidics configuration
– Optical configuration
– Electronic configuration
MIFlowCytData presentation
MIFlowCytData presentation
• Data presentation:
– Flow plots• Label both axis• Fluorophore and antibody• Scale obvious
– Total events
MIFlowCytData presentation
MIFlowCytData presentation
• Data presentation:
– Flow plots• Label both axis• Fluorophore and antibody• Scale obvious
– Total events
CyTOF: mass -cytometry
Bendall et al, Science, 2011
Child & Family Research InstituteChild & Family Research Institute
