Validation of an immunophenotyping method to characterise of lymphocyte subpopulations in Crl:WI(Han) rat spleen by FACSVerse flow cytometer

Abstract

OECD guideline 443 describes the requirements for an Extended One-Generation Reproductive Toxicity Study in the rat. One cohort assesses reproductive/developmental endpoints, including investigation of pre- and postnatally induced immunotoxic effects. The aim of this study was to develop and validate flow cytometry assays to characterise rat spleen lymphocyte subpopulations, and to ensure that accurate and relevant data was obtained that satisfied the guideline requirements. Homogenised spleen samples from Crl:WI(Han) rats were processed for lymphocyte subpopulation evaluation using the FACSVerse flow cytometer. Samples were stained with T-, B-, and NK-cell or T-lymphocyte antibody cocktails, and assessments were performed for precision and stability. Staining with the T-, B-, and NK-cell cocktails or T-lymphocyte antibody cocktail resulted in clear separation of the subpopulations; within-run precision was acceptable with CVs of 0.88 to 6.87% and 0.79 to 3.71%, respectively. Between-run precision was acceptable for samples stored unstained in RPMI with CVs of 0.62 to 18.88% for the lymphocyte subset assay and 0.52 to 11.89% for the T-lymphocyte assay. Minimal carryover was observed of both stained cells into unstained cells and all cells into PBS. Immunophenotyping of spleen lymphocyte subpopulations using T-, B-, and NK-cell and T-lymphocyte antibody cocktails by FACSVerse flow cytometry is reliable and precise assays. Samples should be analysed on the day of collection where possible. Alternatively, samples are stable when stored in RPMI unstained for up to 2 days with daily replacement of RPMI or unstained in stain buffer for up to 1 day, prior to staining and analysis.