Entific St. Louis, MO, USA) was constituted in DMSO as 50 /ml and frozen at -37 until used. The caspase 3 inhibitor, DEVDfmk and substrate DEVD-afc were purchased from Biovision (Palo Alto, CA). ResiquimodMedChemExpress Resiquimod culture media (RPMI 1640), antibiotics, trypsin-EDTA, and other reagents were purchased from Sigma scientific (St. Louis, MO, USA). Cell lines Human prostate adenocarcinoma (PC3) was a generous donation from Rambaugh-Goodwin Cancer Research Institute, Plantation FL. Cell Culture To assess the chemosensitivity of human prostate cancer cells to single and combination treatment with genistein (Gn) and -lapachone (bLap), cells were sub-cultured under 5 CO2 at 37 for 48 hrs to reach 80?0 confluence. All cells were grown and maintained as monolayers in 25 m2 tissue culture flasks (Sigma Scientific, St. louis, MO, USA) in RPMI 1640 containing 15 mM HEPES, and supplemented with 0.45 glucose (w/w), 5.0 FBS and 100 U. mL-1 penicillin + 100 mg. ml-1 streptomycin.Page 6 of(page number not for citation purposes)Cancer Cell International 2004, 4:http://www.cancerci.com/content/4/1/The cells were then harvested by gentle scraping with a cell scraper. The cell suspensions were then grown at a density of 2.5 ?103 cells/well in 24-well microtiter plates (MTP) for 36 hr to allow adherence. The supernatants were discarded and the agents (Gn or bLap) were added over a range of 5 cytotoxic concentrations in single and combination treatments. In preliminary studies with bL1-8, the IC50 ranged between PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26080418 1.8? for a number of cells. Therefore in the present studies, 2.0 (bL2) was used in the combination studies with varying concentrations of genistein. All treatments were in triplicates. Dicoumarol was added to the cells and incubated for 4 hr before treatment with either genistein or -lapachone alone or in combination. All plates were then incubated at 37 in a humidified atmosphere of 5 CO2 in air for a maximum of 72 hr. At 12, 24 and 36 hr of incubation, 100 of the supernatant from each well was gently aspirated and replenished with 100 of fresh media. The supernatants were stored at -37 until assayed for lactate dehydrogenase (LDH) enzyme activity. At 36 hr incubation, the cells were harvested by trypsinization with trypsin-EDTA, and processed for post-treatment metabolic activity using cell viability and apoptotic assays as described.A. Cell Viability Assays A1.1 MTS assay MTS assay depends on mitochondrial enzyme reduction of MTS solution to detect and determine cell viability. The MTS cell proliferation assay is a colorimetric method for determining the number of viable cells in proliferation. It is composed of solutions of a tetrazolium compound [3(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate; PES). MTS is bioreduced by the cells into formazan product that is soluble in cell culture medium. Following cell culture as described above, 100 of cells were harvested from each treatment group and added to a 96 MTP followed by addition of 20 of MTS (2.5 mg/mL: Sigma Chemical Co) stock solution to each well. After 2 hr incubation under standard conditions of 5 CO2 and 37 , the purple formazan product (indicative of reduction of MTS) was visible. The absorbance was read on Multiskan biochromatic automated microplate reader (Multiskan, DC) at 490 nm. The signal generated (color intensity) is directly proportional to the number of viable (meta.