Single cell suspensions (total 5000 cells in 2 ml) in Dulbecco's modified Eagle's medium/F-12 containing 5 mg/mL insulin, 0.5 mg/mL hydrocortisone, 2%B277 (Invitrogen Ltd., Paisley, Scotland), and 20 ng/mL epidermal growth factor were seeded into ultra-low attachment plates (Corning, Lowell, MA) and incubated for 7-10 days in presence of 20% O2 and 5% CO2 at 37°C.
After transfection of reprogramming factors, they were seeded on gelatin coated flasks (BD), cultured in MEF conditioned human embryonic stem cell medium (HES Medium) containing 20% Knock-out serum replacement (KSR) [14] and 8 ng/ml of FGF2.
To determine the effect of StAR overexperssion on the mRNA expression of inflammatory factors, RAECs were seeded in 6-well plates and treated with PA (200 μM) at different time (0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h) following with 48 h infection.
Immortalized human renal tubular epithelial cells (HKC) were cultured at 37 °C with humidified atmosphere of 5%% CO2 in DMEM/F12 supplemented with 10%% fetal bovine serum (FBS) and 2 ng/mL mouse epidermal growth factor and were seeded into 96-well culture plates (Corning, Schiphol-Rijk, The Netherlands) for 24 h.
A mammosphere assay was also performed in DMEM/F-12 media containing 5 mg ml−1 insulin, 0.5 mg ml−1 hydrocortisone, 2%B277, and 20 ng ml−1 epidermal growth factor, and cells were seeded into six-well ultra-low attachment plates.
In the growth factor treatments, 1×104 cells were seeded as the starting point, and 1×105 cells in the case of inhibitor treatments.
After 16 h of serum and growth-factor privation, ECFC (3 × 10 cells/well) were seeded on growth factor-reduced Matrigel (200 μl) (BD Biosciences) in EBM2 medium and cultured for 18 h at 37°C with 5% CO2.
To assess the response to exogenous growth factors, 1.5 × 105 fibroblasts were seeded in 6-well plates.
Cells transfected with luciferase reporter plasmids containing respective binding-sites of various transcriptional factors and Renilla-TK construct were seeded into 48-well plates.
For experiments with cells cultured under the conditions without growth factors, neurospheres were dissociated and cells were seeded at a density of 5 × 10 cells cm−2 onto PDL and laminin-coated 96-well plates in the culture medium containing 20 ng mL−1 EGF and 10 ng mL−1 bFGF.
