Percp cy5.5 conjugated anti cd11b

Approximately 10⁶ cells were incubated for 15 min with the Fc-receptor-blocking antibodies anti-CD16/anti-CD32 (BD Biosciences Pharmingen, San Diego, CA, USA) and with a Zombie Aqua™ viability dye (BioLegend, San Diego, CA, USA). After washing with PBS + 2% fetal calf serum (FCS), the cells were stained for 30 min with the fluorescein isothiocyanate (FITC)-conjugated anti-CD3 (cat. No. 11-0031-81, eBioscience, San Diego, CA, USA), phycoerythrin (PE)-conjugated anti-Ly6G (cat. No. 12-9668-82, eBioscience, San Diego, CA, USA), allophycocyanin (APC)-conjugated anti-F4/80 (cat. No. 17-4801-80, eBioscience, San Diego, CA, USA) and PERCP-Cy5.5-conjugated anti-CD11b (cat. No. 45-0112-80, eBioscience, San Diego, CA, USA). Next, cells were washed twice and fixed with 0.37% formaldehyde in PBS. Cells were analyzed on a BD LSR Fortessa X20 with DIVA software (BD, Franklin Lakes, NJ, USA, v9.0). Results were further analyzed with the FlowJo software (BD, Franklin Lakes, NJ, USA, v10.0). Flow cytometry plots showing the gating strategy for cellular identification are depicted in Supplementary Figure S2.

Hepatic immune cells were isolated as we described previously [33 (link)]. Collected immune cells were stained with APC-CY7-conjugated anti-CD45 (#A15395, eBioscience, San Diego, CA, USA), PerCP-CY5.5-conjugated anti-CD11b (#45-0112-80, eBioscience, San Diego, CA, USA), PE-conjugated anti-F4/80(#MF48004-3, eBioscience, San Diego, CA, USA), BV421-conjugated anti-Ly-6G (#9668-82, eBioscience, San Diego, CA, USA) and APC-conjugated anti-Ly-6C (#17-5932-82, eBioscience, San Diego, CA, USA). The purity of these isolated immune cells was over 90%.