Establishing Orbital Fibroblast Cultures

The primary cultures of orbital fibroblasts were established according to our previous study [16 (link)]. Briefly, the orbital tissues were minced aseptically in phosphate-buffered saline (PBS, pH 7.3) and then incubated with a sterile solution containing 0.5% collagenase and dispase (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) for 24 hours in an incubator filled with an atmosphere of 5% CO₂ and kept at 37°C. The mixture of digested orbital tissues was pelleted by centrifugation at 1,000 ×g and then resuspended in Dulbecco's Modified Eagle's Medium (DMEM, purchased from Gibco Life Technologies, Gaithersburg, MD, USA) containing 10% fetal bovine serum (FBS) and a cocktail of antibiotics (Biological Industries, Kibbutz Beit Haemek, Israel), which was composed of 100 U/mL penicillin G and 100 μg/mL streptomycin sulfate (Biological Industries, Kibbutz Beit Haemek, Israel). The cultured orbital fibroblasts were used between the 3rd and 5th passages and the cell cultures at the same passage number were used for the same set of experiments.