KGN Cell Culture and Stimulation

Procedures have recently been described [2 (link)]. The patented KGN cell line was obtained from RIKEN BioResource Center [4 (link)] with permission by T. Yanase. KGN cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/Ham’s F12 medium (Life Technologies, Paisley, UK) supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL) (Biochrom, Berlin, Germany) and 10% fetal calf serum (FCS) (Capricorn Scientific, Ebsdorfergrund, Germany) at 37 °C and with 5% CO₂. For stimulation experiments, 20 µM Trolox (Santa Cruz Biotechnology, Dallas, TX, USA) or 100 µM/1 mM H₂O₂ (Sigma-Aldrich, St. Louis, MO, USA) was diluted in DMEM/Ham’s F12 medium (Life Technologies; colorless medium without phenol red was used for live cell fluorescence imaging to reduce background autofluorescence).