Rpmi 1640 culture medium

MCF-7 breast adenocarcinoma and NCI-H460 non-small cell lung cancer human cell lines (European Collection of Cell Culture, Salisbury, Wiltshire, UK) were grown in RPMI-1640 culture medium (Lonza, Basel, Switzerland), supplemented with 5% fetal bovine serum (FBS), and cells were maintained at 37 °C in a 5% CO₂ humidified atmosphere.

Lung cell suspensions were prepared after enzymatic digestion of the lungs using digestion buffer, containing DNase I and Collagenase A (Roche Diagnostics), for 30 min. The digestion was stopped by adding fetal calf serum (FCS, Hyclone Laboratories, Logan, USA). The lung pieces were passed through a 70 μm filter and rinsed with 10 mL RPMI. Cells were washed and resuspended in RPMI 1640 culture medium (Lonza, Allendale, USA) supplemented with 10% heat-inactivated FCS and 0.1% penicillin-streptomycin solution (Sigma-Aldrich). Lung cells (4 × 10⁵ cells/well) were cultured in medium with or without 50 μg/mL HDM (Greer Laboratories). The supernatant was harvested after 4 days of culture at 37°C in 5 % CO₂ and stored at −20°C until further analysis (31 (link)).

The human myelomonocytic leukemia cell line U937 (ATCC, Manassas, VA, USA) was grown in RPMI-1640 culture medium (Lonza, Verviers, Belgium) supplemented with 5% fetal calf serum (FCS, PAA Cell Culture Company, Pasching, Austria), 20 mM HEPES, 2 mM L-glutamine, and 100 U/ml penicillin/streptomycin (all from Lonza, Verviers, Belgium). Cells were cultured in 25cm² culture flasks (Greiner Bio-One GmbH, Frickenhansen, Germany) in 10 ml medium at an initial density of 3 × 10⁵ cells/ml and in a humidified atmosphere at 37°C and 5% CO₂. Cell cultures were refreshed every 3-4 days.
Acquired resistance to CHR2863 was induced by exposing U937/WT cells to a starting concentration of 15 nM CHR2863 (IC₁₀) for one week. Then, the concentration of CHR2863 was gradually stepwise increased when cells had adapted to drug increments by exhibiting cell growth comparable to control U937/WT cells. Over the course of CHR2863 increments, two sublines of CHR2863 resistant U937 cells were selected for further detailed characterization; (a) one with a relatively low level of acquired resistance (≈ 14-fold) isolated after 2.5 months when grown in the presence of 200 nM CHR2863 (further designated as U937/CHR2863(200), and (b) another with a high level of CHR2863 resistance (> 250-fold) isolated after 5-6 months when grown in the presence of 5 μM CHR2863 (further designated as U937/CHR2863(5μM) cells).

Dr. Y Shimada (Kyoto University) gifted human ESCC cell lineage, which were cultured in RPMI 1640 culture medium (Lonza, Switzerland) containing 1% penicillin/streptomycin and 10% fetal bovine serum (Gibco, Grand Island, NY, USA) in an incubator (37 °C, 5% CO₂). The ESCC specimens were acquired from Shanghai Outdo Biotech Co. Ltd., and the patients were first informed and then their consent was taken. The investigation was authorized by the Research Ethics Board of Peking University Cancer Hospital.

Human epidermoid carcinoma (A431) cells and murine macrophages (RAW 264.7) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS) (Bodinco, Alkmaar, the Netherlands), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM l-glutamine (all from Lonza). Human umbilical vein endothelial cells (HUVECs) were isolated as described in [25 (link)] and maintained in EndoGro-LS complete culture medium (Merck Millipore, Billerica, MA, USA). HUVECs were grown in Primaria cell culture flasks (Corning Life Sciences, Tewksbury, MA, USA). Human perihilar cholangiocarcinoma (SK-ChA-1) cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 culture medium (Lonza) supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM l-glutamine, and 143 µM β-mercaptoethanol. All cells were maintained at standard culture conditions (37 °C, 5% CO₂, 95% air, humidified atmosphere).