Glutagro

All cells were maintained at 37 °C with 5% CO₂. DM1 fibroblasts and wild-type fibroblasts were maintained in growth medium [1× MEM (Corning), 10% FBS (Sigma), 1% MEM non-essential amino acids (Corning), 1% antibiotic/antimycotic solution (Corning), and 1% Glutagro (Corning)], and cells were treated with compound dissolved in growth medium. Conditional MyoD-fibroblast cell lines (Arandel et al., 2017 ) were grown in growth medium [1× DMEM (Corning), 10% FBS, 1% antibiotic/antimycotic solution, and 1% Glutagro]. For FECD studies, F35T and F20T cells were grown in growth medium [1× OptiMEM-1 (Gibco), 5 ng/ml EGF (Human EGF Cat# J64012), 20 ng/ml NGF (Invitrogen Cat# 13257-019), 20 μg/ml ascorbic acid, 200 mg/l calcium chloride, 0.08% chondroitin sulfate, 50 μg/ml gentamicin, 1% antibiotic/antimycotic solution (Corning), and 8% FBS].

The immortalized human retinal Müller cell line MIO-M1 was obtained from the Department of Cell Biology of the University College, London. Cells and were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5g/L glucose, glutaGRO (Corning Cellgro, Manassas, VA), and 10% fetal bovine serum (FBS) as reported previously (Hollborn et al., 2011 (link); Limb et al., 2002 (link); Ramírez et al., 2016 (link)). It should be noted that the high glucose levels in both control and treated media are higher than that of healthy individuals (normal glucose levels in the body 70 to 130 mg/dL) and may have some influence on the viability of the cultured cells as well as the expression of different genes. However, the MIO-M1 cell line requires higher glucose levels for healthy culture conditions and some reports in the literature show that these high glucose levels in the culture media did not affect the gene expression levels (Matsuzaki et al., 2014 ). In this study, MIO-M1 cells were plated in either 6-well, 24-well or 96-well plates for 24 hours before treatment and kept under normal culture conditions of 37° C and 5% carbon dioxide.