Ciprofloxacin

The human embryonic kidney cell line 293T and the U87MG human glioma cell line were obtained from the American Type Culture Collection (Manassas, VA, USA). 293FT and Lenti-X 293T cells were purchased from Thermo Fisher Scientific (Waltham, MA, USA) and Takara Bio Inc. (Shiga, Japan), respectively. The A549 human non-small cell lung cancer cell line, PANC-1 human pancreatic cancer cell line, NCI-H1299 human metastatic lung cancer cell line, Huh7 human hepatocellular carcinoma cell line, and CSC2 human glioma stem cell line were obtained from various researchers as described previously^{17 (link)–21 (link)}. The 293T, 293FT, Lenti-X 293T, U87MG, PANC-1, and Huh7 cells were cultured in Dulbecco’s modified Eagle medium (DMEM; HyClone - GE Healthcare, Chicago, IL, USA). The A549 and NCI-H1299 cells were cultured in RPMI 1640 medium (HyClone). The CSC2 glioma stem cells were cultured as described previously^{20 (link)}. All media except that used to culture CSC2 were supplemented with 10% fetal bovine serum (HyClone), 1% penicillin/streptomycin (Welgene, Gyeongsan, Republic of Korea), and 10 μg/ml ciprofloxacin (Santa Cruz Biotech, Santa Cruz, CA, USA).

The tissue material was first rinsed with 50% ethanol (Merck, Darmstadt, Germany), followed by phosphate buffer (Invitrogen, Waltham, MA, USA). The necrotic and adipose tissues were then removed from the tissue using a sterile scalpel, and the tumour tissue was cut into 3 mm pieces. Next, samples in the trypsin media were kept at 37 °C to activate the trypsin. Subsequently, the tumour pieces were transferred to a sterile tube with phosphate buffer and centrifuged at 4 °C, 2700 rpm for 7 min. After removing the supernatant, the tumour pieces were placed into the culture medium (MEM) with added 10% FBS (Biochrom, Holliston, MA, USA), 1% antibiotic-antimycotic solution, 10 μg/mL gentamicin sulfate, and 10 μg/mL ciprofloxacin (Santa Cruz Biotechnology, Dallas, TX, USA) to prevent contamination. After 7 days the culture medium was replaced with MEM medium with 10% FBS, supplemented with antibiotics (penicillin 100 U/mL and streptomycin 0.1 mg/mL) (Biochrom, Holliston, MA, USA) and 0.4 μg/mL hydrocortisone (Merck, Darmstadt, Germany). Since we were primarily interested in the effect of TME-derived fibroblasts (CAFs), selection using population overgrowth was performed. The passages of CAFs ranged from 1 to 5.