Leibovitz s l 15 medium

OC cell lines; HTB75 (ATCC, CAOV-3; Adenocarcinoma), HTB161 (ATCC, OVCAR-3; Adenocarcinoma) and HTB76 (ATCC, CAOV-4; Adenocarcinoma) cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (01-055-1A; Biological Industries, Beit HaEmek, Israel) supplemented with 10% fetal bovine serum (FBS) (04-127-1A; Biological Industries), RPMI medium (01-100-1A; Biological Industries), supplemented with 20% FBS, and Leibovitz’s L-15 Medium (01-115-1A; Biological Industries) supplemented with 20% FBS respectively. HaCaT (Keratinocytes, CLS Cell Lines Service GmbH, Eppelheim, Germany; [21 (link)]) skin cells were cultured in DMEM supplemented with 10% FBS. All media were supplemented with 1% Pen-Strep, 1% L-Glutamine and 0.02% plasmocin. Cells were incubated at 37 °C in a humidified atmosphere; HTB75, HTB161 and HaCaT were grown in an environment containing 5% CO₂ and 95% air, while HTB76 cells were grown in air. Induction of the mesenchymal phenotype was done as described in [21 (link)], with modifications: HTB75 and HTB161 were seeded in complete medium 3 days prior to induction. Induction conditions for HTB161 were RPMI medium, including FBS 5% and IL-1β 30 ng/mL, and for HTB75 DMEM medium, including FBS 1%, IL-1β 30 ng/mL and TNFα 100 ng/mL for 48 h. Treatments were applied for 16 h, while maintaining induction conditions.

To analyze SC sensitivity to 1,6-hexanediol at different temperatures, worms grown at 20°C or 25°C were dissected in Leibovitz’s L-15 medium (Biological Industries) supplied with 10% fetal bovine serum (Biological Industries) and 0.01% Tween 20 (complete L-15 medium). Intact gonads were transferred to 1 ml complete L-15 medium in 1.5 ml tubes incubated in 20°C or 25°C water bath for 5 min. After the settlement of the gonads, the supernatant was removed and 1 ml complete L-15 medium containing 5% 1,6-hexanediol was added. After 5 min incubation in 20°C or 25°C water bath, the supernatant was removed and the gonads were fixed with 1 ml 4% paraformaldehyde in PBS for 30 min. After two washes with PBST, gonads were stained with DAPI and mounted on slides with coverslips. Images were captured with a Nikon TS2-FL microscope.