Effects of strain, sex and age on immunophenotyping parameters in the rat and mouse

Abstract

Immunophenotyping of lymphocyte subsets can be a useful addition to toxicological or investigatory studies where there is the potential for effects on the immune system. We have validated assays for rat or mouse lymphocytes using a FACSVerse flow cytometer and FACSuite software, with commercially available antibodies. Whole blood was stained using the following markers: Rat—CD3, CD45RA, CD161a, CD4 and CD8; mouse—CD3e, CD19, NK1.1, CD4 and CD8. This allows detection and quantitation of T cells, B cells, NK cells, T helper cells and T cytotoxic cells. The stopping criteria for analysis were set at 30,000 lymphocytes, although data were considered acceptable if a minimum of 5000 lymphocytes were counted. To maintain the same analysis protocol throughout and across studies, an assay was created for each species in the FACSuite software. Assay functionality uses CS&T beads for performance quality control and assay setup to ensure that laser settings remain consistent and compensation settings are such that any drift in machine performance does not affect results. Blood samples from both sexes and the following ages and strains were analysed: Rats—2, 4 and 7 months, Crl:WI(Han) and 2 months Crl:CD(SD); mice—2–3, 4–6 and 7–8 months, Crl:CD1(ICR). In general, there was a low level of variation within the results for each sex or age group. However, there are strain, sex and age differences that should be considered when evaluating results from immunophenotyping analysis or when planning studies where there may be an immunological effect.