1. Advances In Flow cytometry 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

2. 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

4. Types of controls Instrument set up control Gating controls
PMT voltage settings
Compensation
Gating controls
Isotype controls
FMO controls
Biological controls
Unstimulated samples
Healthy donors

5. Fluorescence Minus One is not always an ideal control
FMO
FMO quadrant copy/pasted
CD4- quadrant setting

7. No FRET with proper panel of conjugated Abs
Gal-Ser tetramer
CD3

9. Flow cytometry of rare populations
Minimal Residual Disease
GFP
PBL stem cells
BM stem cells
Tetramers: Ag specific T cells

10. Rare population & Flow cytometry Practical Considerations
Number of events should be increased for statistical accuracy 200,000-1,000,000
Proper controls
Proper instrument setting

11. Intracellular staining
Cytokine
&
Phospho protein

12. Intracellular staining
Cytokines
Type of stimulator
Time point
Type of protein transport inhibitor
Level of production
Background level
Type of Fix/perm
Color selection

13. Intracellular 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

14. CD4
CD3
IL-4
IL-17
IFN-g
IFN-g

15. Phospho-protein detection
Type of stimulator
Time point
Level of production
Background level
Type of Fix/perm
Color selection

16. B-cell Acute lymphoblastic leukemia cell lines
Phospho-protein
TLR2 ligand Pam2CSK
Pre B-cell leukemia cell lines
Phosphorylation of NFkB & degradation of IkB

17. Time 5
Time 0
Time 10
Time 20

18. Dose response curve and phospho-protein kinetic analysis
TLR1
TLR-2
TLR-6
Dose response curve
and phospho-protein
kinetic analysis
TLR1
TLR-2
TLR-6
TLR1
TLR-2
TLR-6

19. B-cell acute lymphoblastic leukemia:
IL-7R expression
Healthy control
Ig Light chain
B-ALL relapse
IL-7Ra

20. YB-1 in acute lymphoblastic leukemia
Interruption of host immune system recognition
By HLA-DO up-regulation/decreasing
Ag-presentation
Prevention of apoptosis
by Survivin
YB-1
Blocking the effect of cytotoxic drugs
Through IL-7 signaling and multi-drug resistance -1 (MDR-1) up-regulation

21. Novel IL-7 signaling pathway in B-cell ALL Phosphorylation of YB-1
pYB-1
No IL-7
IL-7 stimulated
CD19

22. Treg 6-color staining CD3-PECY7, CD4-PerCPCY5
Treg 6-color staining CD3-PECY7, CD4-PerCPCY5.5, CD25-PE, CD127-FITC, CD45RA-V450, FOXP3-AF647

23. Immunophenotyping on preserved whole blood sample using
Cyto-Chex BCT tube

24. B-cell Immunophenotyping

25. CD19+ (Pacific Blue) gated cells
Peripheral Blood
Bone marrow
IgD-FITC
IgD-FITC
CD10-APC
CD27-PE
CD27-PE

26. Whole blood Treg phenotyping using eBioscience Kit CD3-APCH7, CD4-AF700, CD25-PE, Helios-AF488, FOXP3-V450
CD3+CD4+ gated T cells
Cyto-Chex tube
EDTA tube

27. Treg phenotyping and drug monitoring of an unique IPEX case CD3-AF488, CD4-PerCPCy5.5, CD25-PE, CD127-BV421, FOXP3-AF647 HD
IPEX
CD25-PE
FOXP3-AF647

28. CD25-PE HD
IPEX
CD127-BV421

29. Immuno-supressor Monitoring detection of surface CD69 up-regulation upon CD2 activation (IPEX patient)
CD3+CD4+ gated T cells
Un-stimulated
Stimulated
Corticosteroid
Sirolimus

30. The Minimum Information about Flow cytometry Experiment (MIFlowCyt)
Ref: Cytometry part A
73A:
2008

31. Simplify cross-platform comparison and integration,
MIFlowCyt
Simplify cross-platform comparison and integration,

32. MIFlowCyt Experimental overview: Purpose/goal hypothesis
Experiment variables
Conclusions
Quality control

33. MIFlowCyt Flow sample (specimen): Material Source/organism/location
Treatment
Reagent/analyte/detector/reporter

34. MIFlowCyt Data presentation
Experimental and Sample Information:
Experimental design
Sample preparation reagents
Fluorescent reagents
Vendor
Catalog number
Clone

35. MIFlowCyt Data presentation
Data acquisition:
Instrument configuration
Instrument identification
Fluidics configuration
Optical configuration
Electronic configuration

36. MIFlowCyt Data presentation
Flow plots
Label both axis
Fluorophore and antibody
Scale obvious
Total events

37. MIFlowCyt Data presentation
Flow plots
Label both axis
Fluorophore and antibody
Scale obvious
Total events

38. CyTOF: mass -cytometry

40. Bendall et al, Science, 2011

42. Child & Family Research Institute