Concanavalin a cona

Concanavalin A (con A) was purchased from Alfa Aesar, Erlenbachweg 2, Kandel, (Germany). Naloxone hydrochloride was purchased from abcam Biotechnology, Cambridge, (UK). Alanine amino transferase (ALT), aspartate amino transferase (AST), albumin, and bilirubin were purchased from Bio-Med diagnostics, (USA). Reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and thiobarbituritic acid reactive substance (TBARS) assay kits were purchased from Bio-diagnostic, (Egypt). Rat interferon-γ (IFN-γ) enzyme-linked immunosorbent assay (ELISA) kit, rat TNF-α ELISA kit, rat IL-6 ELISA kit and rat IL-1β ELISA kit were purchased from MyBioSource, San Diego, California, (USA), Cusabio, Houston, (USA), R&D Systems, McKinley Place NE, Minneapolis, (USA) and MyBioSource, San Diego, California, (USA), respectively. An antibody raised against Nuclear factor kappa B (NF-κB p65), c-Jun N-terminal kinase (JNK), Nuclear factor erythroid 2 (Nrf2), Heme oxygenase-1 (HO-1), TLR4 and β-actin were purchased from Thermo Fisher Scientific, Waltham, (USA). Goat Anti-Rabbit HRP linked IgG and Goat Anti-Mouse HRP linked IgG were obtained from abcam Biotechnology, Cambridge, (UK).

Three extracellular substrates were tested for their ability to support cell viability and to modulate the morphology of individual cells and cell clusters. Glass bottom 47 mm diameter Petri dishes (MatTek, Ashland, MA, USA) were coated with 300 µL of 100 µg/mL Poly-L-lysine (PLL, Sigma-Aldrich), 15 µg/mL Concanavalin A (Con-A, eBioscience) or 80 µg/mL Laminin (LM; Gibco, Grand Island, NY, USA) at room temperature (25 °C) for 1 h. Uncoated dishes served as controls. The supernatant was then removed, and the dishes were washed 3× with PBS. All liquid was removed from the dish and placed in a 25 °C incubator overnight. Cell cultures were maintained in a 25 °C incubator and fresh Schneider’s medium was added to cell cultures after 24 h. Cells were assessed for morphology and viability at 0, 24 and 48 h. Brightfield images of cell cultures were taken to assess morphology of individual cells and RPC clusters. Viable cells on each substrate were tested after 24 h and 48 h using the Colorimetric Cell Viability Kit III XTT (Invitrogen). Potential reductions in cell viability over time were assessed by comparing XTT absorbances with values obtained from assays of samples of newly-dissected cells (n > 15 eye-brain complexes, isolated as described). All absorbance values were normalized against those on uncoated Petri dishes.

MSCs or AhR-activated MSCs were co-cultured with splenocytes using a cell–cell contact approach. Different ratios of MSCs or AhR-activated MSCs and splenocytes (0, 1:50, 1:10, and 1:5) were loaded to a 24-well, round-bottom plate. After the adherence of MSCs or AhR-activated MSCs to the plate wall, CFSE-labeled splenocytes at a concentration of 1 × 10⁶/well were added to the 24-well culture plate and concanavalin A (ConA) (eBioscience) was mixed at a concentration of 3 μg/mL to stimulate the proliferation of splenocytes. The CFSE-labeled splenocytes alone with no ConA were used as the negative control. These cells were cultured for 3 d, and the proliferation of cells was characterized by FACS.