Tnf α

Primary porcine RPE cells were treated with 50 µg/mL octopus’s ommochrome extract as well as 1 µg/mL LPS (Sigma-Aldrich), 10 µg/mL Poly I:C (Tocris BioScience, Bristol, UK) or 50 ng/mL TNFα (Tocris BioScience) for one, three and seven days [37 (link)]. Supernatants were collected for 24 h and interleukin 6 and interleukin 8 secretion was investigated with porcine IL-6 and IL-8 DuoSet ELISA Kit as described in the user manual.

All experiments were approved by the Vanderbilt University Institutional Animal Care and Use Committee and were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Six-week old male C57BL/6 mice were procured from Charles Rivers (Wilmington, MA). Mice received a 2 μl intravitreal injection of TNFα (50ng/ml) plus vehicle (0.1% DMSO in PBS) or INCA-6 (25 μM; Tocris; Minneapolis, MN). Six hrs later, mice were anesthetized with ketamine and xylazine and perfused at physiological pressure (between 100 and 122 mmHg for mice) with 0.9% saline for 6 min, followed by FITC-conjugated concanavalin-A (40 μg/ml in 2.5 ml PBS, Vector Laboratories; Burlingame, CA). Residual non-adherent leukocytes were washed out using an additional 6 min saline perfusion. Retinas were dissected in 4% paraformaldehyde, flat-mounted, and imaged with an AX70 upright scope (Olympus) and DP71 digital camera (Olympus) at 4× magnification, then lumenal leukocytes were manually counted by two masked observers. Retinal leukocyte counts for an entire retina were averaged and reported as retinal leukocytes per mm². Each treatment arm consisted of at least 5 retinas.

Keratinocytes were cultured in 96-well plates at a concentration of 1 × 10⁴ cells/well for 12 h. Cells were then treated with PBS containing TNF-α (2 ng/mL) or Wnt3a (50 ng/mL; Tocris, Bristol, UK) alone, or in combination in KBG gold medium for 24, 48 and 72 h at 37 °C in 5% CO₂. Ten micro liters of 3-(4,5-dimethlthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) (5 mg/mL dissolved in PBS; Sigma, St. Lois, MO, USA) was added to each well, and the plates were incubated at 37 °C for 2 h. The supernatant was discarded and 200 μL of dimethyl sulfoxide (DMSO; Sigma, St. Lois, MO, USA) was added to dissolve the blue insoluble MTT formazan produced by mitochondrial succinate dehydrogenase. The absorbance was measured spectrophotometrically at 570 nm. Experiments were repeated three times and the data were expressed as the means ± SD.

Insulin (Sigma): 10⁻⁷M for 30 min. C₂-Ceramide/C₆-ceramide/C₂-Dihydroceramide (BIOMOL in Enzo Life Science, Farmingdale, NY, USA): dissolved in DMSO (vehicle), then diluted into serum-free DMEM at the indicated concentrations and briefly sonicated, concentration used in the experiment is 50 μM, in serum-free medium in 0.1% BSA RIA Grade (Sigma) for 48 h. TNF-α (Sigma): 100 ng/ml for 72 h. FumonisinB1 (Cayman Chemical Company, Ann Arbor, MA, USA): 100 μM, 30 min before the stimulation with TNF-α. LY294002 (Calbiochem, San Diego, CA, USA): 30 μM, 30 min before the stimulation with insulin, C₂-ceramide or C₂-Dihydroceramide. SB202190, H-89 (Calbiochem): 10 μM, 30 min before the stimulation with insulin, C₂-ceramide or C₂-Dihydroceramide. GSK650394 (Tocris, Bristol, UK): 103 nM, 60 min before the TNF-α stimulation. Z-DEVD-fmk (BD Pharmingen, Milan, Italy): 20 μM, 60 min before the TNF-α stimulation.