Mouse blood monocytes: Standardizing their identification and analysis using CD115

Monocytes have been used to assess immune dysfunction and disease. While mouse models are a useful longitudinal analog, few researchers have assessed changes in mouse monocytes. The purpose of this study was to provide recommendations for the sample processing and flow cytometric analysis of mouse blood monocytes. Blood was drawn in a non-lethal manner from CD-1 male mice to be used in three experiments. Experiment 1 compared commonly used mouse monocyte markers. Experiment 2 compared the stability of CD115 expression after immediate (0 h) and delayed (2 and 4 h) processing following blood collection under various experimental conditions (laser strength, anticoagulant, and storage temp.). Experiment 3 compared the consistency of CD115(+) monocyte and subset concentrations using decreasing (40, 20, 10 and 5 mu L) volumes of blood. In experiment 1, >95% of CD115(+) events co-expressed CD11b; >85% co-expressed CD14. 70% of CD14(+) and 50% of CD11b(+) events co-expressed CD115. In experiment 2, CD115 expression decreased by 33% between 0 and 4 h when stored at room temperature. Blood treated with EDTA and refrigerated maintained CD115 stability. In experiment 3, calculated concentrations for total monocyte events varied by <10% when 40,20 and 10 mu L of blood were stained. While CD115 staining provides the most distinct monocyte population, it is important to treat blood with EDTA and refrigerate if sample processing will be delayed over 2 h. Collectively, the findings of the present study outline important considerations that must be addressed when examining mouse monocytes in small, non-lethal blood samples. (C) 2011 Elsevier B.V. All rights reserved.