Pe vio770 conjugate anti mouse cd11b clone m1 70

Mouse primary liver cells were isolated following a previously established method (You et al. 2008 (link)). In brief, the mouse liver was perfused with Hank's balanced salt solution followed by a digestion buffer containing 0.2% collagenase (Type IV, Sigma). Single cell suspensions were filtered through a 100 µm cell strainer and centrifuged at 50 g for 3 min. Hepatocytes were in the pellet, and the cells in the supernatant were collected and further centrifuged in 35% percoll (Sigma, Germany) to obtain liver NPCs. Red blood cells were lysed with red blood cell lysing buffer. Liver NPCs were stained with fluorochrome-conjugated antibodies for multi-color fluorescent-activated cell sorting (FACS) analysis, including APC Cyanine7conjugated anti-mouse CD45 (clone 30F11, #130-105-506; Miltenyi Research Inc. San Diego, CA), APC conjugated anti-mouse F4/80 (clone BM8, #17-4801-82, eBioscience), PE-vio770 conjugate anti-mouse CD11b (clone M1/70, #25-0112-82, eBioscience), PE-conjugated anti-mouse Ly6G (clone RB6-8C5, #561084, BD Biosciences) and FITC-conjugated anti-mouse Ly6C (clone AL21, #553104, BD Biosciences, San Jose, CA). BD FACSCanto II flow cytometer was used and the software FlowJo (V10) was utilized to analyze data.

To prevent non-specific binding, freshly isolated liver NPCs were treated with mouse Fc receptor blockers. Fluorochrome-conjugated antibodies were then added to the cells and incubated. These antibodies included APC-conjugated anti-mouse F4/80 (clone BM8, #17-4801-82, eBioscience, San Diego, CA, USA), FITC-conjugated anti-mouse Ly6C (clone AL21, #553104, BD Biosciences, San Jose, CA, USA), PE-vio770 conjugate anti-mouse CD11b (clone M1/70, #25-0112-82, eBioscience), APC Cyanine7 conjugated anti-mouse CD45 (clone 30F11, # 103116, Biolegend, San Diego, CA, USA), and PE-conjugated anti-mouse Ly6G (clone 1A8, #127608, Biolegend). Data analysis was performed using the BD FACSCanto II flow cytometer and the FlowJo (V10) software.

Freshly isolated LNPCs were incubated with mouse Fc receptor blocker to prevent non-specific binding. Then, the cells were incubated with various staining antibodies, including APC Cyanine7conjugated anti-mouse CD45 (clone 30F11, #130-105-506; Miltenyi Research Inc. San Diego, CA), PE-vio770 conjugate anti-mouse CD11b (clone M1/70, #25-0112-82, eBioscience), APC conjugated anti-mouse F4/80 (clone BM8, #17-4801-82, eBioscience), FITC-conjugated anti-mouse Ly6C (clone AL21, #553104, BD Biosciences, San Jose, CA) or PE-conjugated anti-mouse Ly6G (clone RB6-8C5, #561084, BD Biosciences).