Ki 67 clone 11f6

The following antibodies were purchased from BioLegend: CD16/32 (clone 93), CD45 (clone 30-F11), CD3 (clone 145-2C11), CD4 (clone GK1.5), CD8α (clone 53-6.7), CD25 (clone PC61), CD62L (clone MEL-14), CD69 (clone H1.2F3), PD-1 (clone 29 F.1A12), CD44 (clone IM7), Granzyme B (clone QA16A02), LAG-3 (clone C9B7W), TIM-3 (clone RMT3-23), TCR γ/δ (clone GL3), NK1.1 (clone PK136), and TNF-α (clone MP6-XT22), LAP (clone TW7-20B9), IL-10 (clone JES5-16E3), and Ki-67 (clone 11F6). FOXP3 (clone FJK-16s) and TCR beta (clone H57-597) were purchased from eBioscience.

To enrich LECs from hepatic NPCs we thawed frozen samples in RPMI containing 10% Human Serum AB (Gemini Bio-products, West Sacramento, CA) and 1% DNASE (MP Biomedicals, Santa Ana, CA). Cells were washed 2x with PBS containing 2% FBS (Atlas Biologicals, Fort Collins, CO) and stained with antibodies against CD45 (clone HI30), CD31 (PECAM1, clone WM59), Podoplanin (clone NC-08) and CD146 (clone P1H12) from Biolegend (San Diego, CA), and CD68 (clone KP1) from abcam (San Francisco, CA). Cells from either Non-diseased, NASH or HCV were sorted using an aria Fusion sorter (BD Biosciences, Franklin lakes, NJ) and enriched LECs were sorted into RPMI containing 50% Human Serum AB. Enriched LECs from each condition were pooled for single cell sequencing as described below. Flow cytometric analysis of human liver LECs: isolated liver NPCs were stained with Fixable viability dye 510 (BD Biosciences) and stained with the above surface antibodies. Following surface staining cells were fixed and permeabilized (Thermo Fischer, Waltham, MA) and stained for Ki67 (clone 11F6) (Biolegend). Flow cytometry was performed using a BD FACSCanto II instrument and was acquired with BD FACSDiva software (BD Biosciences). Analysis ws performed using FlowJo 10 (Treestar, Woodburn, OR).