Discrepant phenotyping of monocytes based on CX3CR1 using fluorescent reporters and antibodies

Abstract

Abstract Monocytes as well as downstream macrophages and dendritic cells are essential players of the immune system fulfilling key roles in homeostasis as well as in inflammatory conditions. Conventionally, driven by studies in reporter models, mouse monocytes are divided into a classical and a non-classical subset based on their inversely correlating surface expression of Ly6C and CX3CR1. Here, we analysed the expression of CX3CR1 by flow cytometry using several validated fluorochrome-coupled CX3CR1 antibodies and compared them with the reporter gene signal of a Cx3cr1GFP reporter mouse strain as well as of tamoxifen-inducible Cx3cr1 reporter mice. Although we were able to validate the specificity of several fluorochrome-coupled CX3CR1 flow cytometry antibodies, mouse Ly6Chigh classical and Ly6Clow non-classical monocytes showed no differences in CX3CR1 expression levels in peripheral blood and spleen, when stained with these antibodies. To the contrary, in reporter mice, we were able to reproduce the inverse correlation of CX3CR1 reporter gene signal and Ly6C surface expression. As determined by qPCR, the Cx3cr1 mRNA expression correlated with the GFP-reporter gene expression as quantified by flow cytometry. In conclusion, our data suggest that there is differential transcription, but not surface expression of CX3CR1 between classical and non-classical monocytes, which limits the suitability of CX3CR1 for phenotyping monocyte subsets by antibody staining.