Whole blood peripheral blood leukocyte immunophenotyping
Peripheral blood leukocyte assay is performed at 6 weeks of age (Mice were fed on Mouse Breeder Diet (5021, Labdiet) from weaning)
Non-fasted unanaesthetised mice are restrained and blood is collected into EDTA coated tubes via the tail.
Whole blood is stained with a titrated cocktail of antibodies including CD45, TCRab, TCRgd, CD161, CD4, CD8, CD19, CD25, CD44 and CD62L.
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 and B cells are reported as percentage of leukocytes. CD4+ CD25+ regulatory alpha beta T cells, CD4+ CD44hi CD62Llo alpha beta T cells and CD8+ CD44hi CD62Llo alpha beta T cells 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.