Sb203580

Five corneal rims samples from normal donor corneas (three for 22 years old and two for 25 years old) were used to the HCECs culture and cell culture was performed by Wuhan PriCells Biomedical Technology Co., Ltd. (Wuhan, China) according to described protocols [8 (link)]. Briefly, Descemet’s membrane with intact endothelium was carefully dissected in small strips and then incubated in OptiMEM-I supplemented with 10 % FBS overnight to stabilize the cells before culture. After centrifugation, the strips were incubated in 0.02 % EDTA solution at 37 °C for 1 hour and cells were resuspended in culture medium containing OptiMEM-I, 8 % FBS, 5 ng/mL EGF, 20 ng/mL NGF, 100 g/mL pituitary extract, 20 g/mL ascorbic acid, 200 mg/L calcium chloride, 0.08 % chondroitin sulfate, 50 g/mL gentamicin, and antibiotic/antimycotic solution diluted 1/100. Cultures were then incubated at 37 °C in a 5 % carbon dioxide, humidified atmosphere.
The cultured HCECs were then subjected to various H₂O₂ concentrations (0 to 100 μM) for variable time periods (0 minutes, 1 hours, 2 hours and 6 hours) at 37 °C. The cells with no H₂O₂ treatment were used as controls. In addition, HCECs were also treated with SB203580 (10 μM) (Cell Signaling Technology, Inc., Danvers, MA) and the cells with no SB203580 treatment were used as controls.

Suspension Jurkat cells were grown in DMEM enriched with 10% v/v FBS, L-glutamine, penicillin, and streptomycin at 37 °C in 5% CO₂. For SB203580 inhibitor pretreatment, Jurkat cells (10⁷) were suspended in 5 ml enriched DMEM and SB203580 (Cell Signaling Technology) was added from 10 mM stock to a final concentration of 10 μM. Equal volume of DMSO was added to control samples. The cells were then incubated at 37 °C for 2 h until treatment with pervanadate and calyculin (PVA/CA). Cells were centrifuged at 300g for 5 min at 4 °C and washed twice with PBS. After the last wash, the pellet was resuspended in 5 ml enriched DMEM, and PVA/CA was added to a final concentration of 100 and 0.1 μM, respectively. Control samples received equal volumes of PBS and DMSO vehicles, respectively. Both groups were incubated at 37 °C for 20 min. TCR activation was achieved as previously described with modifications (21 (link)). Jurkat cells (5 × 10⁶) were resuspended in 1 ml enriched media and treated with 5 μg/ml anti-CD3ε and CD28 antibodies and 30 μg/ml goat anti-mouse IgG to cross-link the primary antibodies. The cells were then incubated for indicated times at 37 °C, after which they were harvested.