Skbr3 htb 30

Raji (CCL-86^™), ARH-77 (CRL-1621^™), Daudi (CCL-213^™) and SK-BR-3 (HTB-30^™) cells were purchased from ATCC (Manassas, VA, USA). Raji, ARH-77 and Daudi were propagated in RPMI with 10% fetal bovine serum (FCS) (both Sigma-Aldrich, St. Louis, MO, USA) with 4 mM glutamine, sodium pyruvate and penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C in humidified atmosphere with 5% CO₂ and diluted three times a week to be kept below 1 × 10⁶ cells/mL. ARH-77 cells were detached with a cell-scraper (TPP, Trasadingen, Switzerland). SK-BR-3 cells were kept in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) with 10% FBS with 4 mM glutamine and penicillin/streptomycin and passaged twice a week by Trypsinization: the cells in a T75 flask were rinsed twice with 12 mL pre-warmed PBS, detached with 3 mL 0.1% Trypsin/0.05 M EDTA (Sigma-Aldrich, St. Louis, MO, USA) for 5 min at 37 °C, at 5% CO₂ in humidified atmosphere, and collected in 9 mL DMEM with all additives with a centrifugation at 300× g, 5 min at RT before resuspension in fresh complete medium.

Primary human breast epithelial HMEC cells (CC-2551, Lonza, Basel, Switzerland) and immortalized breast epithelial 184A1 cells (a kind gift from A/Prof. Darren Shafren, the University of Newcastle) were cultured in mammary epithelial basal medium (MEBM) supplemented with bovine pituitary extract (BPE) (0.4%), human epidermal growth factor (hEGF) (0.1%), hydrocortisone (0.1%), GA-1000 (0.1%), and insulin (0.1%) (Lonza). MCF7 (HTB-22, American Type Culture Collection (ATCC), Manassas VA, USA), T-47D (HTB-133, ATCC), MDA-MB-231 (HTB-26, ATCC), and SKBR3 (HTB-30, ATCC; a kind gift from A/Prof. Darren Shafren, the University of Newcastle) breast cancer cell lines were cultured in RPMI-1640 (GE Healthcare, Chicago, IL, USA) supplemented with 10% FBS (Sigma–Aldrich, St. Louis, MO, USA) and 2 mM L-glutamine (GE Healthcare). All cells were maintained at 37 °C with 5% CO₂ and used within four years of purchase from ATCC or Lonza, or authenticated using the GenePrint 10 System (Promega, Madison, WI, USA) as per the manufacturer’s instructions, and DNA fragments were detected by the Australian Genome Research Facility (AGRF) (Melbourne, VIC, Australia).

The human breast cancer cell lines SKBR3 (HTB-30) and BT474 (HTB-20) were obtained from American Type Culture Collection. Monolayer cultures of the cells were maintained in DMEM/F12 medium with 10% fetal bovine serum. The cell lines overexpressed the HER2/c-erb-2 (HER2) gene product. The JIMT-1 cell line that was established from a breast cancer patient who clinically demonstrated resistance to trastuzumab was purchased from German Collection of Microorganisms and Cell Cultures. The JIMT-1 cell line also showed HER2 overexpression [20 (link)]. Both SKBR3 and JIMT-1 lacked expressions of estrogen and progesterone receptors (ER/PR) and BT474 expresses ER in addition to HER2 receptor.