Product: PE anti-mouse CD45

Catalog No.: 103106

Jeffrey Fites, Postdoc University of Wisconsin-Madison

Cost Effective and Robust Method of Staining Intravascular Leukocytes in vivo with anti-CD45
Overall:	Product Quality:	Ease of Use:
Anti-CD45-PE is an effective reagent for intravenous administration to stain blood leukocytes and is inexpensive enough to make this method cost effective.

Experimental Design

Application:	Flow cytometry
Cells used:	peripheral blood and lung leukocytes
Brief Protocol:	Adapted from Anderson et al. 2014 (http://www.ncbi.nlm.nih.gov/pubmed/24385150, DOI: 10.1038/nprot.2014.005). 2 ug of anti-CD45-PE delivered intravenously (retro-orbital or tail vein) 3 minutes before euthanizing mice. Lungs and peripheral blood collected and processed. Leukocytes stained ex vivo later with anti-CD45.2 and myeloid surface markers.
Results Summary:	All peripheral blood leukocytes stained with administered anti-CD45. Naive lung shows that the resident alveolar macrophages are in negative gate while neutrophils and inflammatory monocytes are in positive gate (both of which almost exclusively in capillaries in healthy lung).
Additional Notes:	This technique requires administration of micrograms of antibody per mouse potentially costing the researcher lots of money in antibodies. The PE conjugate from BioLegend works better than biotin (due to some off-target staining of streptavadin conjugates) and is very reasonably priced. PE withstands mechanical and enzymatic processing of lung, but these steps were complete as much as possible in the dark to limit loss of fluorescence. Later replicates of this experiment with infected lungs showed greater resolution when lungs where rinsed immediately in a large volume of PBS and processed in double the typical volume of digest buffer because excess antibody tends to cross-over and stain cells not in capillaries. (This is more noticeable for DCs and macrophages). Method has not been attempted in conjunction with ICS.

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Publication Status This data has not been published.

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