Advances In Flow cytometry Amina Kariminia Ph.D.

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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

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

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

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

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

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

Intracellular staining Cytokine & Phospho protein

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

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

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

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

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

Time 5 Time 0 Time 10 Time 20

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

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

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

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

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

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

B-cell Immunophenotyping

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

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

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

CD25-PE HD IPEX CD127-BV421

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

The Minimum Information about Flow cytometry Experiment (MIFlowCyt) Ref: Cytometry part A 73A:926-030 2008

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

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

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

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

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

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

MIFlowCyt 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 Institute