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MOD Immunology (Weiss)

MOD Immunology (Weiss). 21th January – 26th January 2013. Experiment 1: Goals. Quantification of cell count Cell vitality via MTT-assay Measuring of LD50 Basophile Release Assay 50% of max β - Hex release. Experiment 1: Quantification of cells.

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MOD Immunology (Weiss)

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  1. MOD Immunology (Weiss) 21th January – 26th January 2013

  2. Experiment 1: Goals • Quantification of cell count • Cell vitality via MTT-assay • Measuring of LD50 • Basophile Release Assay • 50% of max β-Hex release

  3. Experiment 1: Quantification of cells Quantification of cell count via „Neubauer Zählkammer (improved“): • 11 minor chambers: 137 cellsAverage value: 12,45→ 1 Major Chamber: 12,45 * 25 = 311 cells • Cell count per milliliter:1 mm3 := 1 µl; volume of 1 major chamber= 0,1 µl311 cells/0,1 µl * 10 = 3113,63 cells/µl→ 3 * 106 cells/ml • 100µl/ well (Dilution 1:2,5) 3 * 104 cells/ml /2,5= 120 * 103 cells/well

  4. Experiment 1: Procedure • Seed Cells • Cell Lysis via dilution series of Triton X-100 (2% - 0,004%) • Centrifugation • Transfer supernatant • Measurment of the effect

  5. Experiment 1: Effect • β-Hexosaminidase • in granulocytes • degrades gangliosides (4-NAG) • Indication via change of color • → Detection of cell lysis • MTT • Reduction in Mitos and ER • Electro-chemic potential essential • → Detection of cell viability

  6. Experiment 1: β-Hexosaminidase EC50: 0,0143 % → 50% of maximal degranulation

  7. Experiment 1: MTT-Assay

  8. Experiment 2: Goals • Calibration via FACS (Tuesday) • Adjust sensibility of scanners • Spectral compensation • Immuno-phenotyping of allergic vs. non-allergic mice and analysis of cell count via Cyflogic-Software • Comparision of results of all groups

  9. Experiment 2: FACS

  10. Experiment 2: Cell count of leucocytes Analysis of cell counts via Cyflogic-Software Calculation: 10000 St.beads pro 20µl 1278 St.beads measured by FACS 10000/1278 = 7,8 Leucos: 14500 * 7,8 = 113458 113458 / 20 = 5673 cells/µl

  11. Experiment 2: Cell count of CD4+/CD8+

  12. Experiment 2: Cell count of extrathymic T-cells CD4+/CD8+ CD4-/CD8+ CD4+/CD8-

  13. Experiment 2: Cell count of eosinophiles

  14. Experiment 2: Cell count of B-cells and monocytes B-cells Monocytes

  15. Experiment 2: Cell count of neutrophiles

  16. Experiment 2: Total amount of cells 5673 2092 445 114 1712 250 1272

  17. Experiment 2: Statistical analysis of Leucos and CD4 Overall amount of Leucos is higher in non-allergic mice even though it should be in reverse The CD4+ T-Helper cells are increased in allergic mice. In both cases is no significance

  18. Experiment 2: Statistical analysis of CD8/CD14 Total amounts of CD8+ CTLs and CD14+ monocytes are increased in non-allergic mice. The results are not significant.

  19. Experiment 2: Statistical analysis of CD19/Eos The amount of B-cells in non-allergic mice is slightly increased, while the total amount of eosinophiles is higher in allergic mice. Both results are not significant.

  20. Experiment 2: Statistical analysis of neutrophiles Again, in the non-allergic mice the amount of leucocytes (here neutrophiles) are increased in relation to allergic mice, although not significantly.

  21. Experiment 2: Comparision with other groups (overall)

  22. Experiment 3: Goals • Comparision of IgE status of allergic vs. non-allergic mice • Immuno-phenotyping of allergic vs. non-allergic mice and analysis of CD200R upregulation via Cyflogic-Software (Basophiles) • Comparision of results of all groups

  23. Experiment 3: Basophile Activation Assay • Analysis of cell ligated IgE via Basophile Activation Assay • In sensitive mice allergen Phl p 1 binds to specific IgE on basophils, crosslinking IgE • Activation of basophile → Degranulation • After Activation: Upregulation of CD200R on Basophiles, specific marker • Sensitive vs. Naive Mice • Positive Controll via Anti- IgE • Buffer only as negative Controll

  24. Experiment 3: Basophile Activation Assay CD19-/IgE+ CD4-/IgE+ Lymphogate and Basophile_1 and Basophile_2 Naive Mouse #3: Buffer only, neg. Controll Naive Mouse #3: Anti- IgE, pos.Controll

  25. Experiment 3: Basophile Activation Assay Naive Mouse #3: Phl p 1 stimulated

  26. Experiment 3: Basophile Activation Assay Sensitive Mouse #3: Buffer only, neg. Controll Sensitive Mouse #3: Anti- IgE, pos.Controll

  27. Experiment 3: Basophile Activation Assay Sensitive Mouse #3: Phl p 1 stimulated

  28. Experiment 3: Basophile Activation Assay SI is always above 1, which means that a reaction took place. A comparision between the average SI of allergic vs. Non-allergic mice shows a significant difference

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