Insulin transferrin selenium 100x

Different types of cells were used in the current study for different purposes. Michigan Cancer Foundation-7 (MCF-7) breast cancer cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (with l-glutamine and high glucose, Gibco), supplemented with 10 %V/V fetal bovine serum (FBS) (Canada origin, ThermoFisher) and 1%V/V penicillin-streptomycin (10,000 U/mL, ThermoFisher) until 70% confluent. C2C12 myoblast cells were grown in the same DMEM, supplemented with 10%V/V heat-inactivated FBS (HI-FBS) (Canadian origin) and 1%V/V penicillin-streptomycin. For differentiation purposes, these cells were cultured in DMEM supplemented with 2%V/V of horse serum (ThermoFisher) and 1%V/V penicillin-streptomycin and 0.1% insulin (Insulin-Transferrin-Selenium, 100X, ThermoFisher, Catalogue number 41400045). SH-SY5Y neuroblastoma cells were cultured in DMEM/F-12 (ThermoFisher, with l-glutamine) medium supplemented with 10% HI-FBS and 1% penicillin-streptomycin. For differentiation of these cells, DMEM/F12 was supplemented with 1% heat-inactivated FBS, 1% N2 supplement, and 1 μM retinoic acid. Red fluorescent protein–tagged human umbilical vein endothelial cells (HUVECs) were grown in EBM-2 medium. Osteoblast-like cells from Saos-2 osteosarcoma cell line were cultured in McCoy's medium (ThermoFisher, with l-glutamine) supplemented with 15% FBS and 1% penicillin-streptomycin.

Immortalized human podocytes, wild-type (WT) and SMPDL3b overexpressors (OE) were cultured on collagen-coated dishes and differentiated in RPMI-1640 medium containing 10% FBS (Sigma-Aldrich) and 5% penicillin/streptomycin (Biowest). Briefly, cells were propagated at 33°C in 1% insulin-transferrin-selenium 100x (Gibco, USA) containing media on T25 flasks and then thermoshifted for differentiation for 14 days at 37°C. A single dose of 8Gy was delivered from an RS2000 X-ray irradiator (225 kV) according to the manufacturer's specifications (Rad Source Technologies, Suwanee, GA, USA). The dose rate was adjusted to 265 cGy/min. Cells were then irradiated (8Gy) and treatment was stopped by removing the media and adding cold saline solution at the proper time points. For ROS scavenging and NOX1/4 inhibition, cells were treated with 100 μM of N-acetylcysteine (NAC) and 10 μM GKT137831 dissolved in DMSO and PBS respectively for 2 hours prior to irradiation. An equal quantity of DMSO or PBS was added to the control samples.