Antibiotic and antimycotic solution

iPSC clumps in DMEM/F12 (Gibco) containing 20% KSR (Gibco), 2 mM GlutaMax (Gibco), 1 × 10⁻⁴ M NEAA (Gibco), 1 × 10⁻⁴ M 2-ME (Sigma), and 1 × antibiotic and antimycotic solution (Sigma) were transferred to petri dishes. After 8-day floating culture, embryoid bodies (EBs) were transferred onto ECL (Millipore)-coated slide glasses, and incubated for another 8 days. After incubation, the cells were fixed with 4% paraformaldehyde in PBS.

Myelogenous leukemia cell line K562 was cultured with RPMI 1640 culture medium, supplemented with 10% fetal bovine serum and 1% antibiotic and antimycotic solution (Sigma, USA), in a cell incubator (Bionex, Model-VS-9160C, Korea) filled with 5% CO₂ at 37 °C and 95% humidity. The culture medium was changed every 48 hours. Well cultured cells were filled into 1.5 ml EP tubes which were fixed onto the motion bar of a Sine Wave Generator (PASCO, USA) (see supplementary Figure S1), which oscillated the samples at frequencies 0, 10, 100, 500 and 800 Hz, within a 10 volt amplitude input, for different durations (1, 2, 4, 8, 12 and 16 mins). The oscillated cells were stained with Trypan Blue and measured using hemocytometry. A batch of cells vibrated for 4 mins was also cultured for 1, 3 and 7 days separately and the death rates were also measured by the same method.

Ethanol (99.80%), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Muller-Hinton agar (MHA), Muller-Hinton broth (MHB), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), and fetal bovine serum (FBS) were received from HiMedia, Mumbai, India. Calcium nitrate tetrahydrate, orthophosphoric acid, gallic acid, catechin, rutin, Dulbecco’s modified Eagle’s medium (DMEM), antibiotic and antimycotic solution, lipid peroxidation kit, Griess reagent, caspases kits (3/7, 8, and 9), antioxidant enzyme kits (SOD, CAT, and GSH), ELISA kits (TNF-α and IL-6), primer sequences (TNF-α, IL-6, iNOS, COX-2, and β-actin), dichloro-dihydro-fluorescein diacetate (DCFH-DA), TRI reagent, rhodamine 123, and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich, Bengaluru, India. The live/dead dual staining kit was purchased from Thermo Fisher Scientific and iScript One-Step RT-PCR kit with SYBR green was obtained from Bio-Rad, Bengaluru, India.

A total of 10 uteri from mares in early luteal phase of the estrous cycle were used. The equine epithelial and stromal cells were isolated following the methodology recently described [22 (link)]. Cells were cultured at 38°C in a humidified atmosphere of 5% CO₂. The culture medium was Dulbecco's modified Eagle's medium/nutrient F-12 Ham (DMEM/Ham's F-12; Sigma-Aldrich, Madison, USA; D8900) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich, Madison, USA; #C6278) and antibiotic and antimycotic solution (Sigma-Aldrich, Madison, USA; #A5955); it was changed every 2 to 3 days. After reaching 90 to 95% confluence (5 or 7 days of the incubation of stromal or epithelial cells, resp.), cells were trypsinized [22 (link)].Further, cells were seeded at a density of 5 × 10⁵ viable cells/mL for epithelial cells and 2 × 10⁵ viable cells/mL of stromal cells in 24 or 96-well plates, regarding the experiment. Both cell types viability were over 90%.The cell culture homogeneity was evaluated using immunofluorescent staining for epithelial and stromal cell specific markers (cytokeratin, vimentin, resp.) as described before [22 (link)]. The epithelial and stromal cell homogeneity was around 97%.

Luteal tissue isolation followed the methodology described before [28 (link)]. Briefly, CL explants were minced into small pieces and 40 mg of tissue was washed three times with a sterile phosphate buffer solution (PBS) containing gentamicin (50 μg/μL) and placed into culture tubes containing 1 mL Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium (D/F medium; 1 : 1 [v/v], D-8900, Sigma) and supplemented with 0.1% BSA and 1% antibiotic and antimycotic solution (Sigma, A5955). Tissue explants were preincubated on a shaker at 37.0°C with 5% CO₂ in air for 1.5 h and then medium was replaced with fresh DMEM supplemented with 0.1% BSA and antibiotics and antimycotic.

The present study utilized two metastatic PCa cell lines from the American Type Culture Collection (ATCC): PC-3 and DU145. These cells were cultured in MEM medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic and antimycotic solution (Sigma Co., St. Louis, MO, USA). The cultures were incubated at 37 °C with 5% CO₂, and the culture medium was changed every two days. The PC-3 and DU145 cell lines were certified (Supplementary Figure S1).