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

Journal of Leukocyte Biology: An Official Publication of the Reticuloendothelial Society

Ruairi W. Lynch^1¹ 

[1]MRC Centre for Inflammation Research University of Edinburgh Edinburgh United Kingdom^1

关键词: endothelial cell;    flow cytometry;    Kupffer cell;   

DOI  :  10.1002/JLB.1TA0517-169R

学科分类：生理学

来源: Federation of American Societies for Experimental Biology

【 摘 要 】

Multicolor flow cytometry and cell sorting are powerful immunologic tools for the study of hepatic mϕ, yet there is no consensus on the optimal method to prepare liver homogenates for these analyses. Using a combination of mϕ and endothelial cell reporter mice, flow cytometry, and confocal imaging, we have shown that conventional flow‐cytometric strategies for identification of Kupffer cells (KCs) leads to inclusion of a significant proportion of CD31^hi 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 endothelial cell‐associated genes by KCs compared with other tissue‐resident mϕ. Our studies also revealed significant and specific loss of KCs among leukocytes with commonly used isolation methods that led to enrichment of proliferating and monocyte‐derived mϕ. Hence, we present an optimal method to generate high yields of liver myeloid cells without bias for cell type or contamination with endothelial cells.

【 授权许可】

CC BY