Tc lab tek chamber slides

2500 Vybrant CM-Dil-labeled HSC-3 cells and 5000 Vybrant DiO-labeled Mfs were co-cultured in TC Lab-Tek Chamber slides (Thermo Fisher Scientific, MA, USA). The cells were allowed to attach o/n in normal growth medium. Thereafter cells were washed with PBS, and medium changed to SF Optimem (Life Technologies, CA, USA). The following experimental groups were created (n = 4): co-cultures of HSC-3 cells and Mfs (M1 Mf, M2 Mf, R848 Mf) were incubated with either DMSO (1:20 000), 0.3 mM Amiloride (Sigma-Aldrich Co.LLC, MO, USA), 3 mM Amiloride, 5 μM NF-κB inhibitor BAY 11-7082 (MerckMillipore, MA, USA) or 3 mM Amiloride plus 5 μM BAY 11-7082. Also monocultures of HSC-3 cells and Mfs were incubated with the same experimental molecules. Incubation was continued for up to 7 days and monitored once every day with the Evos FL Cell Imaging System (Life Technologies, CA, USA).

Fresh-frozen tissue sections were cut onto Superfrost/Plus slides (Thermo Fischer Scientific, Waltham, MA, USA). A negative control was conducted for each run. For immunofluorescence, cells were grown in eight-well TC Lab-Tek Chamber Slides (Thermo Fischer Scientific). The slides were incubated with the primary antibody overnight at 4 C. Intracellular markers were stained with the primary antibody, followed by antimouse IgG conjugated with Alexa Fluor 488 and/or antirabbit IgG conjugated with Alexa Fluor 594 (Invitrogen). The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were recorded using a Leica DFC310 FX microscope.

VybrantCM-Dil labeled HSC-3 cells (3000 cells) with our without Vybrant DiO-labeled M1-, M2- or R848 Mfs (6000 cells) were grown in 8-well TC Lab-Tek Chamber Slides (Thermo Fischer Scientific, MA, USA). Cells were then washed with PBS, fixed with 4% paraformaldehyde for 10 min at +4°C and permeabilized with methanol at -20°C. Non-specific binding was blocked with normal goat/horse serum diluted 1:50 in 2% BSA/PBS for 30 min at RT. Polyclonal rabbit NF-κB p50 (1:400, NLS, Santa Cruz Biotechnology, TX, USA), polyclonal rabbit NF-κB p65 (1:200, Enzo Life Sciences, NY, USA), monoclonal mouse CD163 (1:100, EDHu-1, AbD Serotec, NC, USA), monoclonal mouse CD68/SR-D1 (1:100, R&D Systems, MN, USA) or monoclonal mouse AE1/AE3 pancytokeratin (1:400, Dako, Glostrup, Denmark) in 1% BSA/PBS were applied and kept for 30 min at 37°C followed by o/n at +4°C. Secondary antibodies were diluted 1:200 in 0.1% BSA/PBS and kept for 1 h at RT in dark. After washing samples were mounted with Shandon Immu-Mount with DAPI (Thermo Fisher Scientific, MA, USA). Images were recorded using a Leica TSC SPS confocal laser microscope and the Leica Application Suite Advanced Fluorescence software.