After sacrifice, the liver was perfused with 5 mL phosphate-buffered saline (PBS) through the portal vein and homogenized. Total liver cells were then resuspended in perfusate buffer containing Hank’s balanced salt solution (HBSS; Ca2+ and Mg2+), collagenase (0.01%), and DNase I (0.001%). After filtration through a 70-μm cell strainer, pelleted cells were resuspended in RPMI and layered with 24% OptiPrep. Subsequent to centrifugation, mononuclear cells (MNCs) were isolated at the 40/60% interface. Cells were washed once with a perfusate
buffer containing HBSS (free Ca2+ and Mg2+), bovine serum albumin (0.25%), and DNase I (0.001%) and supplemented with complete culture media (RPMI; fetal bovine serum [10%], penicillin [100 U/mL], streptomycin [100 μg/mL], selleck kinase inhibitor and L-glutamine [200 mM]). Cell types were determined using microscopy. KC-derived ROS were assayed using the Total ROS Detection Kit (ENZO-51011; Enzo Life Sciences, Inc., Farmingdale, NY). In brief, cell preparations were stimulated using lipopolysaccharide (LPS) MDV3100 (Escherichia coli 0111:B4; Sigma-Aldrich, St. Louis, MO) and incubated for 30 minutes at 37°C. Samples were then washed and cells were resuspended in ROS detection solution, incubated with TruStain FcX (antimouse CD16/32; BioLegend, Inc., San Diego, CA), and stained with F4/80 antibody (Ab; AbD Serotec, Oxford, UK).
Subsequent flow cytometric analysis (FCA) is detailed below. Microspheres MCE (Fluoresbrite YG Microspheres, 1.00 μm; Polysciences, Inc., Warrington, PA) were incubated with a total MNC suspension for 20 minutes at 37°C. Reaction was stopped by the addition of 2 mL of ice-cold PBS. Cell preparations were then washed and incubated with TruStain FcX (antimouse CD16/32; BioLegend)
and stained with F4/80 Ab. Subsequent FCA is detailed below. Cell preparations were stained with CD3-fluorescein isothiocyanate/NK1.1-PerCp and F4/80 clone BM8-PerCP-Cy5.5 Abs (AbD Serotec) for identification of NKTs and KCs, respectively. Cells were incubated at 4°C for 20 minutes, followed by the addition of 1 mL fluorescence-activated cell sorting (FACS) buffer (BioLegend) and centrifugation. Cells were resuspended in a final volume of 100 μL of FACS buffer and analyzed by FCA (BD LSR II; BD Biosceinces, San Jose, CA). Quantification of data was performed using FlowJo 5.6.1. Offspring liver sections, at 3 and 12 months of age, were formalin (10%) fixed and paraffin embedded before sectioning. All sections were then stained with hematoxylin and eosin (H&E) and Masson’s trichrome to assess steatosis, inflammation, and fibrosis. Brunt-Kleiner’s NAS was used to semiquantitatively assess degree of injury by an expert liver pathologist blinded to the identity of the groups.