Characterization of Liver Lymphocytes

Livers perfused with 1X PBS (Thermo Fisher Scientific, Waltham, MA, USA) were digested with 2 mg/mL type IV collagenase (Worthington, NY, USA) to obtain single-cell suspensions and the lymphocytes were enriched by Percoll gradient centrifugation as described previously [11 (link)]. Enriched lymphocytes were stained with fluorochrome-conjugated antibodies and fixable viability stain (BD Biosciences, San Jose, CA, USA), and samples were acquired on a Cytek Aurora Spectral Cytometer (Cytek Biosciences, Bethesda, MD, USA) equipped with five lasers. Data were analyzed using FlowJo software v X.10 (Tree Star, Inc., Ashland, OR, USA) as described previously [11 (link)]. Total numbers were computed by multiplying a proportion of specific populations by the total number of enriched lymphocytes/liver. fluorochrome-conjugated antibodies against CD45 (30-F11), CD3 (17A2), CD4 (RM4-5), CD8 (53-6.7), CD44 (IM7), CD69 (H1.2F3), Tbet (4B10), α₄β₇ (LPAM-1; DATK32) and Ly6C (AL-21) were purchased from BD Biosciences (San Jose, CA, USA). fluorochrome-conjugated antibodies against Foxp3 (FJK-16s), Ki67 (SolA15), NK1.1 (PK136), F4/80 (BM8), CD11b (M1/70), Ly6G (RB6-8C5) and Live/Dead Aqua were purchased form ThermoFisher Scientific (Waltham, MA, USA).