Mycoalert plus mycoplasma detection kit

The mouse mammary adenocarcinoma cell line TS/A [49 (link)] (American Type Culture Collection, Manassas, VA, USA) was cultured in Advanced DMEM (Dulbecco’s Modified Eagle Medium, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5% fetal bovine serum (FBS), 10 mM/μL-glutamine (GlutaMAX) and 1% penicillin–streptomycin (all Thermo Fisher Scientific) in a 5% CO₂ humidified incubator at 37 °C. Cells at 80% confluence were trypsinized using 0.25% trypsin/ethylenediaminetetraacetic acid in Hank’s buffer (Thermo Fisher Scientific), washed with Advanced DMEM with FBS, and collected by centrifugation (470 g, 5 min). The cell line was frequently tested for mycoplasma contamination by MycoAlertTM PLUS Mycoplasma Detection Kit (Lonza, Basel, Switzerland) and was confirmed to be mycoplasma free.

B16F10 murine melanoma cell line (ATCC CRL-6475™) and RAW 264.7 murine macrophages (ATCC TIB-71™) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). RAW 264.7 macrophages were purchased in 2019 and B16F10 cells were authenticated by STR profiling and interspecies contamination test in 2019 (IDEXX BioAnalytics, Ludwigsburg, Germany). The B16F10 cells and RAW 264.7 cells were cultured in Advanced Minimum Essential Medium (AMEM, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and Advanced Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco), respectively. Cell media were supplemented with 5% fetal bovine serum (FBS, Gibco), GlutaMAX (100×, Gibco), 100 I.U./mL penicillin (Sandoz, Barleben, Germany), and 0.05 mg/mL gentamicin (Garamycin, Krka, Novo mesto, Slovenia). Cells were routinely subcultured twice a week with the use of 0.25% Trypsin-EDTA solution (B16F10) or cell scraping (RAW 264.7) for detachment and maintained in a 5% CO₂ humidified incubator at 37 °C. The cells were routinely tested for mycoplasma infection by MycoAlertTM PLUS Mycoplasma Detection Kit (Lonza, Basel, Switzerland) and were mycoplasma free. Doubling times of cells were 16 h for B16F10 cells and 22 h for RAW 264.7, therefore, comparable number of divisions were anticipated after the irradiation.

The human endometrial adenocarcinoma cell line, Ishikawa, was purchased from Sigma, (MO, USA) and validated with Short Tandem Repeat DNA profiling. Ishikawa cells were grown at 37°C in MEM medium supplemented with 5% fetal bovine serum (FBS), L-glutamine, and pencillin/streptomycin in a humidified atmosphere containing 5%CO₂. Everolimus (RAD001) and NVP-BEZ235 were obtained from Selleckchem (Provided by Sapphire Biosciences, NSW, Australia). BD Matrigel^TM was used for the establishment of 3D cultures was obtained from BD Biosciences. Mycoplasma testing (MycoAlert^TM Plus Mycoplasma detection kit, Lonza, MD, USA) was conducted at regular intervals for the quality control of cell culture conditions.

U87MG, T98G, U251MG, MDA-MB-231, PC-3, and H1299 were obtained from ATCC. Melanoma cancer cells VMM39 were from Daniel Gioeli (University of Virginia). GBM cell lines were cultured in MEM media. MDA-MB-231 was in DMEM high glucose media, PC-3 in DMEM low glucose, VMM39 and H1299 in RPMI media 1640. GIC lines G34, G44 and G528 were from Jakub Godlewski (Harvard Medical School) and Ichiro Nakano (University of Alabama). GIC lines JWL-022, JWL-131, JWL-578 and JWL-592 were from Jeongwu Lee (Lerner Research Institute). GICs were cultured in Neurobasal media with N2 and B27 supplements (0.5X), with the addition of human recombinant bFGF and EGF (25ng/ml each; R&D Systems). Short tandem repeat profiling was performed within the last six months to confirm the identity of established cell lines or to confirm the human origin of GIC lines lacking established STR profiles. All cell cultures were screened negative for mycoplasma by MycoAlertTM PLUS Mycoplasma Detection Kit (Lonza).

The human ovarian cancer cells COV318 and COV362 (Sigma, MO, USA) were grown at 37°C in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS), L-glutamine, and pencillin/streptomycin in a humidified atmosphere containing 5% CO₂. Everolimus (RAD001) and NVP-BEZ235 were obtained from Selleckchem (Provided by Sapphire Biosciences, NSW, Australia). Short tandem repeat profiling and mycoplasma testing (MycoAlert^TM Plus Mycoplasma detection kit, Lonza, MD, USA) were conducted at regular intervals for the quality control of cell culture conditions and validation of these cell lines.

The CT26 murine colon carcinoma cells (American Type Culture Collection, Manassas, VA, USA) were cultured in an advanced minimum essential medium (RPMI 1640; Gibco, Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 5% (v/v) foetal bovine serum (FBS; Gibco), 10 mL/L L-glutamine (GlutaMAX; Gibco), 10 mL/L Penicillin–Streptomycin (stock solution, 10,000 U/mL, Gibco) in a 5% CO₂ humidified incubator at 37 °C. The cells were routinely tested and confirmed to be free from mycoplasma infection, using MycoAlertTM PLUS Mycoplasma Detection Kit (Lonza Group Ltd., Basel, Switzerland).