Rpmi f 12

Human GI carcinoid cancer cells (BON), were provided by Drs. B. Mark Evers and Courtney M. Townsend, Jr. (University of Texas Medical Branch, Galveston, TX, USA), and bronchopulmonary carcinoid (H727) cells were purchased from the American Type Culture Collection (Manassas, VA, USA). BON cells were maintained in DMEM/F-12 (Life Technologies, Grand Island, NY, USA) and H727 cells were maintained in RPMI/F-12 (Life Technologies, Grand Island, NY, USA), both as previously described. Both media were supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 100 IU/mL penicillin and 100μg/mL streptomycin (Life Technologies, Grand Island, NY, USA). Both cell lines were incubated in a humidified atmosphere of 5% CO₂ at 37°C. Chrysin was purchased from MP Biomedicals (Solon, OH, USA) and dissolved in dimethyl sulfoxide (DMSO) at a 100mM stock concentration. Aliquots were stored at −80°C and freshly thawed prior to treatment. Cell treatments were conducted by plating cells at sub-confluency and allowing them to adhere overnight. The next day, cells were incubated in fresh medium containing Chrysin (1-100μM) for up to 6 days. DMSO concentrations were equalized across all Chrysin treatment groups (100μM DMSO).

Human gastrointestinal carcinoid cells (BON), were gifted by Drs. Courtney M. Townsend, Jr. of the University of Texas Medical Branch (Galveston, TX, USA) and B. Mark Evers of the University of Kentucky (Lexington, KY, USA). Human bronchopulmonary carcinoid (H727) cells were purchased from the American Type Culture Collection (Manassas, VA, USA). BON and H727 cells were grown in DMEM/F-12 (Life Technologies, Grand Island, NY, USA) and RPMI/F-12 (Life Technologies, Grand Island, NY, USA), respectively, at a 5% CO₂ and 37°C atmosphere. Media was supplemented with 100 IU/mL penicillin, 100µg/mL streptomycin (Life Technologies, Grand Island, NY, USA) and 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA). ABT-888 (Selleck Chemicals, Houston, TX, USA) and dacarbazine (Sigma-Aldrich) were stored in aliquots of 10mM in DMSO at -80°C, and freshly thawed before use. Cells were plated at sub-confluency the day prior to treatment, and then incubated in fresh medium containing ABT-888 (0–10µM) for 24 hours, after which dacarbazine was added (0–1000µM) for 2 additional days. DMSO concentrations were normalized across all treatment groups.