1 / 42

Amina Kariminia Ph.D. Oncology Lab, Child & Family Research Institute,

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

mirari
Download Presentation

Amina Kariminia Ph.D. Oncology Lab, Child & Family Research Institute,

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

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

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

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

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

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

  8. Intracellular staining Cytokine & Phospho protein

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

  10. 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

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

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

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

  14. Time 5 Time 0 Time 10 Time 20

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

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

  17. 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

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

  19. Treg 6-color stainingCD3-PECY7, CD4-PerCPCY5.5, CD25-PE, CD127-FITC, CD45RA-V450, FOXP3-AF647

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

  21. B-cell Immunophenotyping

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

  23. Whole blood Tregphenotyping using eBioscience KitCD3-APCH7, CD4-AF700, CD25-PE, Helios-AF488, FOXP3-V450 CD3+CD4+ gated T cells Cyto-Chex tube EDTA tube

  24. Tregphenotyping and drug monitoring of an unique IPEX caseCD3-AF488, CD4-PerCPCy5.5, CD25-PE, CD127-BV421, FOXP3-AF647 HD IPEX CD25-PE FOXP3-AF647

  25. CD25-PE HD IPEX CD127-BV421

  26. Immuno-supressor Monitoringdetection of surface CD69 up-regulationupon CD2 activation (IPEX patient) CD3+CD4+ gated T cells Un-stimulated Stimulated Corticosteroid Sirolimus

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

  28. MIFlowCyt Simplify cross-platform comparison and integration,

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

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

  31. MIFlowCytData presentation • Experimental and Sample Information: • Experimental design • Sample preparation reagents • Fluorescent reagents • Vendor • Catalog number • Clone

  32. MIFlowCytData presentation • Data acquisition: • Instrument configuration • Instrument identification • Fluidics configuration • Optical configuration • Electronic configuration

  33. MIFlowCytData presentation • Data presentation: • Flow plots • Label both axis • Fluorophore and antibody • Scale obvious • Total events

  34. MIFlowCytData presentation • Data presentation: • Flow plots • Label both axis • Fluorophore and antibody • Scale obvious • Total events

  35. CyTOF: mass -cytometry

  36. Bendall et al, Science, 2011

  37. Child & Family Research Institute

More Related