Cell Culture Protocols for Cancer and Normal Cell Lines

MDA-MB-231 and BT-20 cell lines were obtained from late Dr. Gary Kruh (University of Chicago, Illinois). Human primary epithelial gingival keratinocytes (HPK), and normal colon fibroblast CRL1459 cells were purchased from American Type Culture Collection (ATCC#PCS-200-014, and ATCC#CCD-18Co—CRL-1459, respectively, Manassas, VA, USA). These cells were grown as an adherent monolayer with Dulbecco’s Modified Eagle’s Medium (Corning, Tewksbury, MA, USA) supplemented with 10% fetal bovine serum (Biofluids Technologies, Port Richey, FL, USA), 2 mM of L-glutamine and 100 U/mL of penicillin and 100 µg/mL of streptomycin (Thermo Fisher Scientific, Inc., Waltham, MA, USA), as previously described [42 (link)]. HPKs cells were cultured in dermal cell basal medium (ATCC# PCS-200-030) supplemented with factors from Keratinocyte growth kit (ATCC# PCS-200-040) as per manufacture’s protocol. These cells were cultured in a humidified incubator containing 5% CO₂ at 37 °C. All cells were assessed and confirmed to be free of fungi and mycoplasma. Cells were obtained from frozen stocks and cell passaging (up to P4) was performed at 80% cell confluency trypsin + 2.2 mM EDTA in phosphate-buffered saline (PBS).