Rpmi 1640 medium with l glutamine

Human epithelial ovarian carcinoma cell lines OVCAR-5 (derived from the ascitic fluid of EOC patient without prior treatment; tumor characterized to be platinum-resistant) and OVCAR-4 (derived from the ascitic fluid of platinum-refractory EOC patient) were used to assess the combinatorial effects of minocycline and irinotecan. Authenticated OVCAR-5 and OVCAR-4 cells were obtained from ATCC in 2011 and Fox Chase Cancer Center in 2015, respectively, tested free of mycoplasma contamination using commercially available kits (MycoAlert™, Lonza; Latest date: April 2017), and cultured according to the supplier’s instruction. Authenticated OVCAR-5 and OVCAR-4 were propagated for less than 30 and 4 passages after resuscitation, respectively. No further authentication of the cell line was done by the authors. OVCAR-5 cells were grown in RPMI-1640 medium (with L-glutamine, Cellgro) supplemented with 10 % heat-inactivated fetal calf serum (Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin. OVCAR-4 cells were cultured in equivalent conditions, with the exception of supplementing 0.2 U/mL bovine insulin in the medium, as recommended by the supplier. Both cells lines were maintained in an incubator at 37 °C in an atmosphere of 5% CO₂, passaged and plated at required cell concentrations under sterile conditions.

Candida auris 0390 strain was obtained from the Centers for Disease Control and Prevention Antibiotic Resistance Isolate Bank (CDC, Atlanta, GA, USA).^{28 (link)} This isolate is resistant to amphotericin B, azoles and shows decreased echinocandin sensitivity. Cryopreserved yeast cells stored as glycerol stocks in an ultra-low freezer (set at −80 °C) were propagated by streaking a loopful of yeast cells onto agar plates of yeast-peptone-dextrose (YPD). C. auris 0390 strain was cultured overnight into flasks (150 ml) by inoculating yeast cells in 20 ml of liquid YPD medium at 30°C in an orbital shaker (Thermo Fisher Scientific, Waltham, MA, USA) at 180 rpm. Yeast cells were washed with sterile phosphate-buffered saline (PBS) twice after 18 h incubation and the final inoculum size was adjusted by hemocytometer to 1 × 10⁶ /mL for biofilm formation and testing in RPMI-1640 medium with L-Glutamine (Cellgro, Manassas, VA, USA) and buffered with morpholinepropanesulfonic acid (MOPS) at 165 mM and pH 6.9 (Thermo-Fisher Scientific, Waltham, MA). For simplicity, this medium is referred to as “RPMI” from now on.