Dulbecco s modified eagle s medium dmem

Dulbecco’s Modified Eagle’s Medium (DMEM) was obtained from Wisent Bioproducts (Montreal, QC, Canada). Fetal bovine serum (FBS) and 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) were purchased from Thermo Scientific (Waltham, MA). Δ9-THC (1 mg mL⁻¹ in methanol) was acquired from Cayman Chemical Company (Ann Arbour, MI, USA). CBD was provided by Lupos Biotechnology Inc. (Toronto, ON, Canada). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent, all-trans-retinoic acid, hydrogen peroxide (H₂O₂), Cu(II) chloride, dimethyl sulfoxide (DMSO, 99.9%), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, 99.0%) and ascorbic acid (AA) were purchased from Sigma-Aldrich (Oakville, ON, Canada). All the chemicals used were of analytical grade and used as received.

Human MSCs were isolated from AT as previously described [9 (link), 10 (link)]. Briefly, subcutaneous AT samples were washed with phosphate buffered saline (PBS) (Wisent Inc., St. Bruno, QC) and minced with surgical scissors. The tissue was digested with 0.05% collagenase (Sigma‐Aldrich Corporation, MO, USA) dissolved in Hank’s balanced salt solution (Invitrogen, MA, USA). After 2 h at 37C, the collagenase was neutralized with the addition of fetal bovine serum (FBS), and samples were centrifuged (5 min at 2000 rpm). The pellet was resuspended in complete low glucose Dulbecco’s modified Eagle’s medium (DMEM) (Wisent Inc., St. Bruno, QC), supplemented with 10% MSC-qualified FBS (certified FBS-MSC, Gibco Invitrogen) and 1% penicillin/streptomycin (Wisent Inc., St. Bruno, QC). Digested tissue samples were cultured in T75 flasks at 37 °C in 5% CO₂ (1 g of tissue/flask), and non-adherent cells were washed off two days following isolation. Isolated MSC(AT) were seeded at a density of 4,000 cells/cm² in T75 flasks in complete medium and passaged at 80% confluency. Early and late passage MSC(AT) were defined as ≤ 5 and ≥ 15 passages respectively [11 (link), 12 (link)].

Butyrate and niacin were obtained from Sigma, and Dulbecco's modified Eagle's medium (DMEM) was from Wisent. Phosphate-buffered saline (PBS), MTT, dimethyl sulfoxide (DMSO), amphotericin B, penicillin and streptomycin, 0.25% trypsin +0.02% EDTA, and 0.1% collagenase I were purchased from Solarbio. Epidermal growth factor (EGF) was obtained from Corning. ITS was purchased from ScienCell. Fetal bovine serum (FBS) was from BI, and ROS, T-AOC, and Annexin V-FITC/PI kits were obtained from Jiancheng.

A total of 120 CRC and 115 adjacent non-tumor colorectal specimens were obtained from Jiangsu Province Hospital between June 2014 and June 2016. The Research Ethics Committee of Nanjing Medical University has approved the research, and we obtained written informed consent from all patients. All the samples were obtained surgically and conserved at − 80 °C. Patients were not included in this study if they received any preoperative treatment. For the in vitro experiments, cell lines, including five types of CRC cells (SW480, LoVo, DLD-1, HCT116, HT29) and a intestinal mucosal epithelial cell (NCM460), all were conserved in the laboratory. The cell culture medium consisted mostly of Dulbecco’s modified Eagle’s medium (DMEM), including 100 U/mL penicillin, 100 μg/mL streptomycin and 10% fetal bovine serum (Wisent, Canada). All cells were cultured in a 5% CO2 atmosphere at about 37 °C. We purchased the PI3K inhibitor LY294002 from Cell Signaling Technology (Danvers, MA, USA) and the inhibitor was used to treat CRC cells at 10 μM.

HEK 293T cells (obtained from the American Type Culture Collection [ATCC]) were derived from 293 cells, into which the simian virus 40 T-antigen was inserted. Cf2Th cells (ATCC) are canine thymocytes resistant to SARS-CoV-2 entry and were used as target cells in the virus capture assay. 293T cells and Cf2Th were maintained at 37 °C under 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM) (Wisent, St. Bruno, QC, Canada), supplemented with 5% fetal bovine serum (FBS) (VWR, Radnor, PA, USA) and 100 U/mL penicillin/streptomycin (Wisent).

All chemicals were purchased from Sigma‐Aldrich (Oakville, ON, Canada), unless otherwise specified. Dulbecco's modified Eagle's medium (DMEM), trypsin and antibiotic–antimycotic solution were purchased from Wisent Bioproducts. Other cell culture reagents were obtained from Thermo Fisher Scientific. Skin fibroblasts were derived from skin biopsy following standard procedures. Normal control fibroblasts were kindly provided by Dr Eric Shoubridge and Dr Hana Antonicka (McGill University, Montreal, QC, Canada). All cells were routinely maintained in DMEM supplemented with 10% foetal bovine serum and 1% antibiotic–antimycotic solution at 37°C and 5% CO₂. Cells with passage numbers between 8 and 20 were used in all experiments. For stimulation of oxidative phosphorylation, medium with no glucose but containing galactose (10 mM) was used. For treatment with DHB or UQ₁₀, unless otherwise specified, the chemicals were added into culture media for 1 week before cells were used for analyses.