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Analyzing Immune Response With Flow Cytometry

Analyzing Immune Response With Flow Cytometry. Madelaine Taft-Ferguson (2007-2008). Islet Transplant. Type-1 diabetes: autoimmune disease Targets islet cells Can be cured with islet transplant 88% of islet transplants fail after five years Immune system attacks foreign cells

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Analyzing Immune Response With Flow Cytometry

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  1. Analyzing Immune Response With Flow Cytometry Madelaine Taft-Ferguson (2007-2008)

  2. Islet Transplant • Type-1 diabetes: autoimmune disease • Targets islet cells • Can be cured with islet transplant • 88% of islet transplants fail after five years • Immune system attacks foreign cells • Immune response can be measured by cytokine production

  3. Islet Transplant

  4. Islet Transplant

  5. Islet Transplant

  6. Cytokines • Proteins related to immune response • TNF • Fuad (2007) • IFN • Augustine (2005), Bellisola (2006) • IL-2

  7. First Goal • To determine whether ICS is superior to ELISPOT

  8. ELISPOT ELISPOT plate

  9. ELISPOT Capture antibody ELISPOT plate

  10. ELISPOT Cells added ELISPOT plate

  11. ELISPOT Cytokines released ELISPOT plate

  12. ELISPOT Detection antibody ELISPOT plate

  13. ELISPOT Streptavidin-HRP ELISPOT plate

  14. ICS Cytokine Cell Cytokine Cytokine

  15. ICS Permed Cytokine Cell Cytokine Cytokine

  16. ICS Antibody-conjugated dye

  17. ICS Detected using a flow cytometer

  18. Results: ELISPOT vs. ICS • ICS • All readable • Two days • ELISPOTs • Two readable results out of five • Five days

  19. Second Goal • To modify ICS for use on allo-stimulated human cells • Determine sensitivity • Cytokine peaks

  20. Modified ICS Assay 1 • Extended BFA incubation time for shorter workday, more user-friendly • Cells incubated for 24, 72, 96 hours post-stimulation

  21. Modified ICS Assay 2 • Extended BFA time • Cells incubated for 24, 48, 72 hours post-stimulation • Stimulator:responder ratios 1:1, 3:1, 5:1

  22. BD Bioscience Assay

  23. BD Bioscience Assay

  24. Modified ICS Assay 1 Day 1 Day 3 Day 4

  25. Modified ICS Assay 1 Day 1 Day 3 Day 4

  26. Modified ICS Assay 2 Day 1 Day 2 Day 3

  27. Conclusion • ICS is capable of detecting effect of allo-stimulation on cytokine production • Cells can survive extended incubation times in ICS • TNF and IFN peak within 24 hours, IL-2 not expressed until after 72 hours • 3:1 stimulator:responder ratio is best

  28. Future Work • Further modify ICS • Test correlation suggested by the Fuad study (2007) of TNF with transplant rejection • Investigate possible correlation with IFN, IL-2

  29. Acknowledgements • Dr. Pratima Pakala • Kelly Hire and Diem Ngo • Ms. Lois Fruen • Team Research

  30. Analyzing Immune Response With Flow Cytometry Madelaine Taft-Ferguson (2007-2008)

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