F4 80 antibody

HSCs, liver sinusoidal endothelial cells (LSEC) and hepatocytes (HEP) were isolated for mouse liver and cultured according to validated protocols20 (link)21 (link). Kupffer cells (KC) were isolated by fluorescence-activated cell sorting after collagenase/pronase perfusion, using an F4/80 antibody (Invitrogen, USA). Cell purity and functionality were confirmed on morphology and by quantitative polymerase chain reaction (qPCR) for following marker genes (Supplementary Methods S2): Cyp3a11 (HEP), CD32b (LSEC), desmin and Acta2 (HSC)22 (link). 2 Hours after isolation (4 hours for HEP), cells were washed and either solvent or OCA was added to the medium at concentrations of 0.1, 1 and 10 μM, together with vehicle, 1 mg/mL TGF-β1 (R&D Systems, Wiesbaden-Nordenstadt, Germany), TNF-α or LPS. All cells were collected for molecular analysis 24 h after incubation, except for culture-activated HSC that were further stimulated for 7 days. LX2 cells were provided by Vijay H. Shah (Mayo Clinic, Rochester, NY), originally established by Scott Friedman23 (link).

Infiltration of macrophages was confirmed by immunostaining using F4/80 antibody (eBioscience, San Diego, CA). Immunofluorescence staining of kidney tissues for α-SMA was performed following the previously reports^{20 (link)}. Secondary antibody Alexa Fluor 647 chicken anti-rabbit IgG (H + L) (1:200, Invitrogen, Carlsbad, CA) was used. The slides were observed using a BZ-X700 microscope (original magnification power × 200). The images were randomly acquired, with 8 to 10 high power fields collected for each mouse and then quantified within them.

The murine immortalized macrophages RAW264.7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Camarillo, CA) supplemented with 10% fetal bovine serum (FBS, Gibco, Gaithersburg, MD, USA), penicillin (100 U/ml) and streptomycin (100 μg/ml). Bone marrow-derived macrophages (BMMs) were isolated from the femur and tibia of C57BL/6 mice (8–9 weeks) cultured in DMEM containing 10% FBS, penicillin (100 U/ml) and streptomycin (100 μg/ml) and 10 ng/mL murine M-CSF (Peprotech) for 6 days. Medium was changed every 2-day. Primary mouse KCs were isolated by pronase/collagenase perfusion digestion followed by subsequent density gradient centrifugation, as previously described. Magnetic cells sorting (MACS)-based positive selection using a F4/80 antibody (eBioscience) and or selective adhesion was further employed to purify KCs. RAW264.7 cells, BMMs and KCs were polarized into an M1 or M2 phenotype using 20 ng/ml INF-γ and 100 ng/ml LPS or 20 ng/ml IL-4, 20 ng/ml IL-10, respectively. Cells were primed with 100 ng/ml LPS for 4 h, and subsequently stimulated with 10 μM Nigericin or 5 mM ATP for 2 h to induce pyroptosis.

AMs from BALF were aliquoted into fluorescence-activated cell sorting tubes at densities of up to 1 × 10⁶ cells per 100 μl, then blocked with immunoglobulin G (1 μg IgG/10⁶ cells) for 15 min at room temperature. The cells were stained with propidium iodide (1:500; Immuno Chemistry Technology, USA), the fluorescent inhibitor of active caspase-1 called FAM-YVAD-FMK (1:500; Immuno Chemistry Technology), and F4/80 antibody (1:500; eBioscience, USA). The samples were incubated in the dark with conjugated antibody (5 μl/10⁶ cells) for 30 min at room temperature. Cells were washed twice using the flow cytometry staining buffer, then resuspended in flow cytometry staining buffer (400 µl) for analysis. Isotype control antibody (Immuno Chemistry Technology) was used as a negative control.
To identify AM pyroptosis, gating was based on F4/80-positive cells, allowing analysis of fluorescently labeled active caspase-1 (FLICA) and propidium iodide. In this approach, F4/80 + FLICA + PI + cells appeared in the upper right quadrant of the FLICA-PI plot and were considered to be pyroptotic AMs [29 (link)]. Flow cytometry was conducted using an LSR2 flow cytometer (BD Biosciences), and raw data were analyzed using FlowJo software (TreeStar Corporation, USA).