INTERLABORATORY EVALUATION OF THE QUANTIFICATION OF RAT SPLENIC LYMPHOCYTE SUBTYPES USING IMMUNOFLUORESCENT STAINING AND FLOW CYTOMETRY

Abstract

The quantification of rat splenic lymphocyte populations using immunofluorescent staining and flow cytometry was evaluated in an interlaboratory study involving 6 independent facilities employing a common protocol. The study had three specific aims: (1) to establish baseline values for rat splenic lymphocyte populations, (2) to examine variability in flow cytometry data both within and am ong laboratories, and (3) to evaluate single versus dual cell labeling of T-cell subpopulations, as well as quadrant and histogram analysis procedures. Splenic lymphocytes were enumerated following the lysis of red blood cells (RBC) with ammonium chloride. B-cells (anti-rat kappa light chain; clone OX12) were exam ined using a single im munofluorescent label, whereas T-cells (anti-CD5; OX19), T (T) (anti-CD4; W3/ 25), and T (T) helper h cytotoxic/ su ppressor cyt/ su p (anti-CD8; OX8) cells were examined using both single and dual labeling. Cell recoveries and viabilities reported by each laboratory following the lysis o...