Serial Phenotypic Analysis of Mouse Peripheral Blood Leukocytes

Abstract

Repeated phenotypic analysis of mouse peripheral blood leukocytes over short periods of time (2 weeks) has been difficult because of the very limited volumes of blood available under guidelines of the Institutional Animal Care and Use Committee. The loss of leukocytes and variations among laboratories during conventional flow cytometry sample preparation based on lysing and repeated washing have been limiting factors when measuring multiple parameters in small samples. We describe a method of phenotypic analysis using a no-lyse, no-wash staining technique combined with fluorescent triggering for data collection that can be performed on volumes of 20 muL or less of whole blood per set of markers in one tube. This method allows repeated phenotypic analysis of peripheral whole blood from mice. Fluorescent triggering with anti-CD45-PE/Cy5 antibody allows high-quality phenotypic data to be collected for CD4, CD8, TcR- beta, CD45R (B220), CD11b, and Gr-1 epitopes on leukocytes from mouse peripheral blood without lysis. The markers selected cover the major populations in peripheral mouse blood. Reproducibility and time-course data are presented for sampling periods as long as 4 weeks. Data produced by flow cytometers manufactured by two different companies show well-correlated results. An instrument equipped with a gated amplifier or a photomultiplier tube suitable for Cy7 conjugates could measure additional parameters. Because of interference from unlysed erythrocytes, scatter parameters are not useful for identifying cell populations with this method.

Data on the effects of eIF6 downmodulation on the proportions of innate and adaptive immune system cell subpopulations and on thymocyte maturation

“…Whole intact cells were identified by their FSC/SSC profile while dead cells were excluded from the analysis by gating out the propidium iodide positive population. Living cells were then gated on their SSC/CD45 signal [10] .…”

High levels of eukaryotic Initiation Factor 6 (eIF6) are required for immune system homeostasis and for steering the glycolytic flux of TCR-stimulated CD4+ T cells in both mice and humans

“…Intact cells were identified by their FSC/SSC profile and dead cells were gated out according to their propidium iodide signal. Living cells were then gated through the SSC/CD45 signal (Weaver et al, 2001). The following stainings were performed to identify the different leukocytes populations:…”

Biomarkers in Peripheral Blood Associated with Vascular Injury in Sprague-Dawley Rats Treated with the Phosphodiesterase IV Inhibitors SCH 351591 or SCH 534385

“…Data were collected by triggering on CD45-PE/Cy5 on a Coulter Elite flow cytometer (Beckman Coulter Inc., Hialeah, FL, USA), as previously described (Weaver et al 2002).…”