Whole blood peripheral blood leukocyte immunophenotyping
Peripheral blood leukocyte assay is performed at 16 weeks of age (Mice were fed on Mouse Breeder Diet (5021, Labdiet) from weaning)
Non-fasted mice are terminally anaesthetised and blood is collected into EDTA coated tubes via the retro-orbital sinus. Whole blood is stained with two titrated cocktails of antibodies.
Panel 1 containing CD45, TCRab, TCRgd, CD161, CD4, CD8, CD25, CD44, CD62L and KLRG1.
Panel 2 containing CD45, CD19, IgD, Ly6B, Ly6G, Ly6C, CD11b and I-A/I-E.
Samples are fixed and red blood cells lysed prior to acquisition on a BD LSR II flow cytometer after running automated compensation using compbeads and BD FACSDiva software.
Data is analysed using FlowJo after singlet doublet discrimination, a time gate is used to exclude HTS issues and leukocytes identified with a SSC and CD45 gate. Total T cells, alpha beta T cells, CD4+ alpha beta T cells, CD8+ alpha beta T cells, gamma delta T cells, NKT cells, NK cells, B cells, monocytes, granulocytes and eosinophils are reported as percentage of leukocytes. CD4+ CD25+ regulatory alpha beta T cells, CD4+ CD44hi CD62Llo alpha beta T cells, CD4+ KLRG1+ alpha beta T cells, CD8+ CD44hi CD62Llo alpha beta T cells, CD8+ KLRG1+ alpha beta T cells, KLRG1+ NK cells, IgD+ B cells, Ly6Chi I-A/I-Elo monocytes and Ly6Clo I-A/I-Elo monocytes are reported as percentage of parent. Using the white blood cell count obtained from the haematology analysis absolute cell counts are derived for each population and reported as cells/ul.