Isolation and culture of primary glomerular endothelial cells

Mer-KO mice were first genotypically confirmed by conventional PCR using whole tail lysate (Fig. 1A) [17]. Whole kidneys from healthy WT and Mer-KO mice were then surgically removed under sterile conditions. Primary GECs were isolated and prepared to achieve 95–99% purity at the Cell Biologics company (Chicago, IL, USA). Cells were then aliquoted and stored in liquid nitrogen. Tissue culture flasks and plates were coated with gelatin at 37°C for 1 hr. GECs were cultured in the endothelial basal medium supplied by Cell Biologics. Mer deficiency in GECs was further confirmed by Western Blot (Fig. 1A). For LPS treatment experiments, GECs were cultured in 6-well plates one day before stimulation at a density of 0.5×10⁶ cells per well. Cells were washed once with fresh medium the next day and a range of concentrations of LPS was added. Three days later, cells were harvested for Western blot analysis. Culture supernatants were centrifuged for 20 minutes at 15,000 rpm to remove debris and stored at −80°C.

A. Mouse tail lysate was obtained and genotyping PCR was performed as previously described [17]. Western blotting (WB) analysis of Mer expression on primary GECs was also performed (bottom row). B. Kidney sections (4 μm) obtained from C57BL/6 mice were stained with biotin-conjugated anti-mouse Mer and visualized with PE-streptavidin. Glomerular cell subsets were stained with specific markers as indicated in the figure. C. Kidneys from B6. WT and B6.Mer-KO mice were cut into <1mm pieces and subjected to collagenase I and DNase digestion. After EDTA incubation, single cell suspensions were stained for specific markers. Neutrophils were gated for Gr-1 and CD11b double positivity; macrophages were gated on CD11b⁺ and Gr-1^- populations; glomerular mesangial cells, endothelial cells, and podocytes were gated according to surface antigens, as described in Methods. D. Kidney sections were obtained from WT and nephrotoxic serum (NTS)-injected mice [19] and stained with anti-Mer and anti-CD31 antibodies. Data are representative of two repeats. **p<0.01.