An efficient method to isolate Kupffer cells eliminating endothelial cell contamination and selective bias

Multi-colour flow cytometry and cell-sorting are powerful immunological tools for the study of hepatic macrophages, yet there is no consensus on the optimal method to prepare liver homogenates for these analyses. Using a combination of hepatic macrophages and endothelial cell reporter mice, flow-cytometry and confocal imaging, we have shown that conventional flow-cytometric strategies for identification of Kupffer cells (KC) leads to inclusion of a significant proportion of CD31hi endothelial cells. These cells were present regardless of the method used to prepare cells for flow- cytometry and represented endothelium tightly adhered to remnants of KC membrane. Antibodies to endothelial markers, such as CD31, were vital for their exclusion. This result brings into focus recently published microarray datasets that identify high expression of Cdh5 by KC compared to other tissue-resident hepatic macrophages. Our studies also revealed significant and specific loss of KC among leukocytes with commonly-used isolation methods that led to enrichment of proliferating and monocyte-derived hepatic macrophages. Hence, we present an optimal method to generate high yields of liver myeloid cells without bias for cell type or contamination with endothelial cells.