Tissue culture plates

Human glioblastoma multiforme T98G (ATCC, CRL-1690), U87MG (ATCC, HTB-14) and KJT23I (patient-derived) cells as well as human embryonic kidney HEK293T cells (ATCC, CRL-3216), primary human bronchial fibroblasts NHLF and mouse macrophages MAC (ATCC, CCL-46) were routinely cultivated in standard conditions with DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics: penicillin/streptomycin cocktail as it was described elsewhere^{33 (link)}. For each experiment, cells were harvested with 0.25% Trypsin–EDTA solution (Gibco), counted in a Z2 particle counter (Beckman Coulter), and seeded at an appropriate density into tissue culture plates (Eppendorf).

The Ethics Committee of the Medical Research Council of Hungary (ETT-TUKEB, No. 51323-4/2015/EKU) approved all experiments with human samples and informed consent was obtained from the patients. Samples were collected at the Department of Oncosurgery, Uzsoki Hospital, Budapest, Hungary. Patient data are shown in Supplementary Tables S1, S2. Tumor and normal colon tissue dissected at a distance >5 cm from the tumor were isolated and cut into small pieces (<0.5 cm) in PBS. After extensive washing with PBS three times, tissue pieces were incubated in a digestive mix [DMEM high glucose with 20% FBS, 75 U/mL collagenase type IX (Sigma), and 125 μg/mL dispase type II (Invitrogen, Carlsbad, CA, United States)] for 2 h at 37°C with extensive shaking. After removal of tissue pieces, single cells were then centrifuged at 300 g for 5 min, washed twice in PBS and cultured in tissue culture plates (Eppendorf) in fibroblast medium containing 15% FBS.

The Ethics Committee of the Medical Research Council of Hungary (ETT-TUKEB, No. 51323-4/2015/EKU) approved all experiments with human samples and informed consent was obtained from patients. Samples were collected at the Department of Oncosurgery, Uzsoki Hospital, Budapest, Hungary. Tumor tissues were cut into small pieces (<0.5 cm), and after washing with PBS three times, tissue pieces were incubated in a digestive mix (DMEM high glucose with 20% FBS, 125 µg/mL dispase type II (Invitrogen), 75 U/mL collagenase type II (Merck), 1 h) at 37 °C with shaking. Samples were then centrifuged at 300× g for 5 min to isolate single cells, they were washed twice in PBS and cultured in tissue culture plates (Eppendorf, Vienna, Austria) in DMEM high glucose, 10% FBS, penicillin/streptomycin and glutamine (fibroblast medium). In some experiments 5 nM factor VIIa, 10 ng/mL TGFβ, or 5 ng/mL IL1α were used for 4 days in serum-free medium. Patient data are shown in Table S2.