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Immunological Analysis of Allergic Versus Non-Allergic Mice: Experimental Findings

This study investigates immune responses in allergic and non-allergic mice through various experiments targeting cell quantification, cell vitality, and basophile activation. Key objectives included the determination of cell counts and vitalities using assays like MTT and measuring LD50. Additional experiments focused on FACS calibration for immunophenotyping, specifically analyzing different leukocyte populations, and the comparison of IgE statuses. Statistical analyses highlighted differences in T-Helper cells and B-cell amounts, revealing significant trends despite some non-significant results.

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Immunological Analysis of Allergic Versus Non-Allergic Mice: Experimental Findings

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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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