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

作者：Lynch Ruairi W; Hawley Catherine A; Pellicoro Antonella; Bain Calum C; Iredale John P; Jenkins Stephen J^*

来源：Journal of Leukocyte Biology, 2018, 104(3): 579-586.

DOI：10.1002/JLB.1TA0517-169R

• endothelial cell
• flow cytometry
• Kupffer cell

Multicolor flow cytometry and cell sorting are powerful immunologic tools for the study of hepatic m phi, yet there is no consensus on the optimal method to prepare liver homogenates for these analyses. Using a combination of m phi 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(hi) 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 m phi. 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 m phi. Hence, we present an optimal method to generate high yields of liver myeloid cells without bias for cell type or contamination with endothelial cells.

• 出版日期2018-9