Dmem ham s f 12

Primary ACs were isolated from pooled femoral head articular cartilage samples using collagenase D (1.5 mg/ml, Roche Diagnostics, Indianapolis, USA) in DMEM/Ham’s F-12 (Cellgro, Manassas, USA) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin in a humidified incubator with 5% CO₂ at 37°C. Due to the uneven growth rate and cell density of primary chondrocytes (designated as P0), chondrocytes passaged once (P1) were used for various treatments. Cells were seeded in 12-well tissue culture plates at 1 × 10⁵ cells/well for specific experiments.

MCF10A, MDA-MB-231, T47D, and ZR-75-1 cells were obtained from the American Type Culture Collection (ATCC) and authenticated using short tandem repeat (STR) profiling. No cell lines used in this study were found in the database of commonly misidentified cell lines that is maintained by ICLAC and NCBI Biosample. MCF10A cells were maintained in DMEM/Ham’s F12 (CellGro) supplemented with 5% equine serum (CellGro), 10 mg/mL insulin (Life Technologies), 500 ng/mL hydrocortisone (Sigma-Aldrich), 20 ng/mL EGF (R&D Systems), and 100 ng/mL cholera toxin (Sigma-Aldrich). MDA-MB-231 cells were maintained in DMEM (CellGro) supplemented with 10% fetal bovine serum (FBS) (Gemini). T47D cells were maintained in RPMI 1640 (CellGro) supplemented with 10% FBS (Gemini) and 10 mg/mL insulin (Life Technologies). ZR-75-1 cells were maintained in RPMI 1640 (CellGro) supplemented with 10% FBS (Gemini). DMEM lacking glucose, glutamine, and pyruvate was obtained from CellGro. Cells were passaged for no more than 6 months and routinely assayed for mycoplasma contamination.

MCF10A cells were grown in three-dimensional Matrigel cultures as previously described (16 (link)). Briefly, chambers slides were coated with growth factor-reduced Matrigel (BD Biosciences) and allowed to solidify for 30 minutes. 3×10³ cells suspended in assay media containing 2% Matrigel were overlayed on coated chamber slides. Assay medium contained DMEM/Ham’s F12 (Cellgro) supplemented with 2% equine serum (Cellgro), 10ug/ml insulin (Sigma-Aldrich), 500ng/ml hydrocortisone (Sigma-Aldrich), 5ng/ml EGF (R&D Systems) and 100ng/ml cholera toxin (List Biological Labs). For aspirin studies, acini were allowed to grow for 4 days followed by treatment with aspirin every 2 days. Cells were then fixed and stained with Ki67 and laminin V on day 12 as previously described (16 (link)). Phase contrast images were acquired using the Nikon Eclipse Ti microscope. Fluorescent images were acquired using Zeiss LSM 510 Meta confocal microscope.

MCF10A and MDA-MB-231cells were obtained from the American Type Culture Collection (ATCC) and authenticated using short tandem repeat (STR) profiling. Primary human mammary epithelial cells (HMECs) were obtained as described (42 (link)). MCF10A cells and HMECs were maintained in DMEM/Ham’s F12 (CellGro) supplemented with 5% equine serum (CellGro), 10 mg/mL insulin (Life Technologies), 500 ng/mL hydrocortisone (Sigma-Aldrich), 20 ng/mL EGF (R&D Systems), and 100 ng/mL cholera toxin (Sigma-Aldrich). MDA-MB-231 cells were maintained in DMEM (CellGro) supplemented with 10% fetal bovine serum (FBS) (Gemini). Cells were passaged for no more than 2 months and routinely assayed for mycoplasma contamination (MycoAlert, Lonza).