The cultured ARPE-19 cells were analyzed using flow cytometry. The cells were harvested by treatment with 0.25% trypsin- 0.5% EDTA (Gibco, Carlsbad, CA, USA), washed with 1X PBS, and the cell pellet was resuspended in 2% fetal bovine serum in 1X PBS for 1 h at room temperature.
The identification of surface molecules was carried out by incubating the cells with human monoclonal APC-conjugated anti-toll like receptor 4 (TLR4), FITC-conjugated CD14, and PE-conjugated CD86 antibodies (all from Biolegend, San Diego, CA, USA) for 20 min at 4 °C, according to the manufacturer’s recommendations. Cells were washed with 1X PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich, Inc.) for 20 min at room temperature, washed, and acquired with a BD FACSVerse Flow Cytometer (BD Biosciences, San Jose, CA, USA). The results were analyzed using Flow Jo ver. 10 (Beckton Dickinson, East Rutherford, NJ, USA).
The identification of surface molecules was carried out by incubating the cells with human monoclonal APC-conjugated anti-toll like receptor 4 (TLR4), FITC-conjugated CD14, and PE-conjugated CD86 antibodies (all from Biolegend, San Diego, CA, USA) for 20 min at 4 °C, according to the manufacturer’s recommendations. Cells were washed with 1X PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich, Inc.) for 20 min at room temperature, washed, and acquired with a BD FACSVerse Flow Cytometer (BD Biosciences, San Jose, CA, USA). The results were analyzed using Flow Jo ver. 10 (Beckton Dickinson, East Rutherford, NJ, USA).