L cystine hydrochloride

We used our previously described protocol for isolation of Muller glia (Yi et al., 2007 (link)). Eyes from post-natal day (p) 7–8 pups were enucleated after euthanization and the retinas were carefully isolated then dissociated with scissors in Dulbecco Phosphate Buffer Saline (DPBS). To make single cell suspensions, retinas were incubated in DPBS containing papain (16.5 U/mL; Worthington Biochemical Corp, Lakewood, NJ), L-cystine hydrochloride (2 mg/mL; Sigma-Aldrich, St. Louis, MO) and DNAse (125 U/mL; Sigma-Aldrich) for 30 min at 37 °C. The retina fragments were triturated gently for complete dissociation, followed by the addition of ovo mucoid (1.5 mg/mL; Sigma). The isolated retinal cell suspension was centrifuged (800 rpm for 5 min) then plated in plastic dishes with DMEM containing 10% fetal bovine serum (Thermo Fisher Scientific, Grand Island, NY) for 16 hours. The dishes were shaken to remove non-attached cells and the attached Muller glia were allowed to grow for 7 to 10 days in a T-75 flask at 37°C, 5% CO2, to eliminate any remaining retinal neurons and reach sufficient density. The purity of the Muller glial cultures was determined by their morphology and immunodetection with anti-glutamine synthase antibody (Abcam, Cambridge, MA; 1:1000).

UOK262 cell lines were a gift from Dr. Marston Linehan, National Cancer Center, National Institute of Health.(13 (link)) NCCFH1 was previously developed from the pleural fluid of a patient with metastatic HLRCC.(14 (link)) UOK262 cell line was authenticated through Sanger’s sequencing to confirm the presence of c.1316A>C FH mutation. NCCFH1 cell line was authenticated in the same way to confirm the presence of c.1162 delA FH mutation. All HLRCC cells were maintained in RPMI1640 media (Gibco, cat # 11875–093) with 10% Fetal Bovine Serum (Genesee Scientific). Both cell lines have been tested negative for mycoplasma contamination using a QPCR based method.
For cysteine deprivation experiments, L-glutamine, L-methionine and L-cystine free RPMI1640 media (Sigma, Cat # R7513) were used instead. The media was back supplemented with L-glutamine (Sigma, Cat # G7513) to a final concentration of 2mM, L-methionine (Sigma, Cat # M5308) to a final concentration of 0.1mM, and either without L-cystine supplementation for cysteine deprivation group, or with L-cystine hydrochloride (Sigma, Cat # 57579) supplementation to a final concentration of 0.2mM for the control group. Cysteine deprivation experiments were carried out in the presence of 5% instead of 10% FBS to minimize the effects of cysteine present in the Fetal Bovine Serum.