Goat anti clec4f

Isolated liver immune cells and cultured liver macrophages were collected and counted in an automated cell counters (Cellometer Auto X4, Nexelcom Bioscience) after staining for acridine orange (Sigma-aldrich). Antibodies (details in table S2) including BUV396-anti-CD45 (BD bioscience; # 563791), FITC-anti-Ly6G (Biolegend; #127605), APC/Cy7-anti-F4/80 (Biolegend; #123117), PerCP/Cy5.5-anti-Ly6C (Biolegend; #128011), PE/Cy7-anti-CD31 (Biolegend; #102417), Alexa488-anti-iNOS (Thermo Fisher; #53-5920-82), APC-anti-CD11b (Thermo Fisher; 17-0112-82), PE-anti-Tim4 (Thermo Fisher; 12-5866-82), or goat anti-Clec4f (R&D systems; #AF2784) were incubated with FACS buffer (2% fetal bovine serum, 2 mM EDTA, and sodium azide in PBS) on ice for 30 min. In some experiments, the primary cultures of KCs were incubated with 100 ng/ml of biotinylated LPS (Invivogen, #tlrl-lpsbiot) for 2 hours. Then biotin was detected using PE/Cy7-streptividin (Biolegend; #405206). After surface staining of biotin, in some experiments, internalized biotin-LPS was stained using BV605-streptavidin (Biolegend; #405229) in cells permeabilized using the Intracellular Fixation & Permeabilization Buffer Set (Thermo eBioscience; #88-8824). After washing and resuspension, cells were analyzed on a BD FACS Symphony machine and analyzed by FlowJo software (BD biosciences).