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Mouse blood monocytes: standardizing their identification and analysis using CD115
W L Breslin1, K Strohacker, K C Carpenter
1Laboratory of Integrated Physiology, University of Houston, Houston, TX, United States.
Abstract:
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 (0h) and delayed (2 and 4h) 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 5μL) 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 4h 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 10μL 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 2h. Collectively, the findings of the present study outline important considerations that must be addressed when examining mouse monocytes in small, non-lethal blood samples.
Insights
Properly processing mouse blood monocytes is crucial for accurate immune studies. Refrigerating samples with EDTA and analyzing within 2 hours ensures reliable CD115 expression for flow cytometry analysis.
Area of Science:
- Immunology
- Hematology
- Animal Models
Background:
- Monocytes are key indicators of immune status in disease research.
- Mouse models offer a valuable analog for studying immune responses longitudinally.
- Standardized methods for analyzing mouse monocytes, particularly from small blood volumes, are lacking.
Purpose of the Study:
- To establish optimal protocols for sample processing and flow cytometric analysis of mouse blood monocytes.
- To identify reliable markers for mouse monocyte identification and enumeration.
- To determine the stability of monocyte markers under various storage and processing conditions.
Main Methods:
- Non-lethal blood collection from CD-1 male mice.
- Comparison of monocyte markers (CD115, CD11b, CD14) for population identification.
- Assessment of CD115 expression stability at different time points (0, 2, 4h) and conditions (anticoagulant, temperature).
- Evaluation of monocyte concentration consistency across varying blood volumes (5-40μL).
Main Results:
- CD115, CD11b, and CD14 are effective markers for identifying mouse monocytes, with high co-expression rates observed.
- CD115 expression significantly decreased after 4 hours at room temperature but remained stable when blood was treated with EDTA and refrigerated.
- Accurate monocyte concentrations (<10% variation) were obtained using blood volumes as low as 10μL.
Conclusions:
- CD115 is a reliable marker for mouse monocyte identification in flow cytometry.
- For delayed sample processing (>2h), treating blood with EDTA and refrigeration is essential to maintain CD115 stability.
- These recommendations facilitate consistent and accurate analysis of mouse monocytes from small, non-lethal blood samples.
