Tissue culture flasks

Cancer-associated fibroblasts were differentiated from normal human dermal fibroblasts (Lonza) using four melanoma cell lines: A375 and Hs294T obtained from the American Type Culture Collection and WM1341D and WM9 cell lines purchased from Rockland Immunochemicals, Inc. Fibroblasts were cultured in FBM (Fibroblast Growth Basal Medium, Lonza) cell culture medium (supplemented with FGM™-2 SingleQuots™ from Lonza), whereas melanoma cells were grown in DMEM (Dulbecco’s Modified Eagle Medium) medium containing 4.5 g/l glucose and 1.5 g/l NaHCO₃ supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and antibiotics (10,000 U/ml penicillin, 10 mg/ml streptomycin, 25 µg/ml amphotericin B). Cells were cultured in 25 cm² tissue culture flasks (VWR) at 37 °C in 5%CO₂/95% humidified air and passaged twice a week using 0.25% trypsin/0.05% EDTA solution (IITD PAN, Wroclaw, Poland).

The human colon carcinoma CEA-positive, onalespib-sensitive cell line HT55 was obtained from the European Collection of Authenticated Cell Culture (ECACC [43 (link)]), and the colon adenocarcinoma CEA-positive, onalespib-sensitive cell line SNU1544 was obtained from Korean Cell Line Bank (KCLB [44 (link),45 (link)]). CEA expression level and onalespib sensitivity in both cell lines have been previously assessed [27 (link),46 (link)]. The HT55 cells were cultured in Minimum Essential Medium (MEM) (Biowest, Riverside, MO, USA) supplemented with 20% (v/v) fetal bovine serum (FBS; Sigma Aldrich, St. Louis, MO, USA). The SNU1544 cell line was cultured in RPMI [(Biowest, MO, USA, containing 25 nM N-2-Hydroxyethylpiperazine-N’-2-Ethanesulfonic Acid (HEPES)] supplemented with 10% (v/v) heat-inactivated FBS (Sigma Aldrich, MO, USA). Heat inactivation was done at 56 °C for 30 min. All media were supplemented with antibiotics (100 IU penicillin and 100 µg/mL streptomycin, Biochrom GmbH, Berlin, Germany) and L-glutamine (Biochrom GmbH, Berlin, Germany, 2 mM). Monolayer cultures were grown in tissue culture flasks (VWR, Radnor, PA, USA) and incubated in an atmosphere containing 5% CO₂ at 37 °C. After reaching 75–85% confluency, cell passaging was performed using Trypsin-EDTA (Biochrom GmbH, Germany). All cell lines were cultured less than 3 months after purchase.

CHO Flp-In cells (ATTC) were cultured in DMEM/F12 Media (Gibco) supplemented with 5% (V/V) fetal bovine serum (Gibco), penicillin (100 U/mL), and streptomycin (100 μg/mL). Cells were grown at 37 °C and 5% CO₂ in tissue culture flasks (VWR).