Actor NF-kB results in the secretion of embryonic alkaline phosphatase (SEAP), which is detected 23388095 using Quanti-Blue reagent (Invitrogen). Cells were maintained in RPMI 1640 medium containing 11.11 mM glucose in but no phenol red (GIBCO, Carlsbad, CA) supplemented with 10 heat-inactivated fetal bovine serum (FBS, GIBCO), 1 penicillin (GIBCO), 1 streptomycin (GIBCO) and 50 mM 2-ME (Fisher Scientific, Pittsburgh, PA) at 37uC in a humidified incubator with 5 CO2 and 95 air (e.g., standard culture conditions). Prior to experimentation, the cells were starved for 48 h and subsequently cultured with or without 2-ME and/or FBS at 37uC in a humidified Thermo Scientific CO2 MedChemExpress Gracillin tissue culture incubator (NAPCO Series 8000WJ, Thermo Forma, Marietta, OH) equipped with built-in CO2 and O2 monitors and attached nitrogen and carbon dioxide gas supplies. Carbon dioxide was set to 5 v/v and oxygen to 5 of 18 . The oxygen and carbon dioxide contents of the incubator atmosphere were periodically verified using a Fyrite gas analyzer (Bacharach Inc., New Kensington, PA). For some experiments, cultures were treated for 24 or 48 h with phorbol 12-myristate 13-acetate (PMA, ML 264 custom synthesis SigmaAldrich, St. Louis, MO) at 20 ng/ml to trigger 16985061 THP-1 cells to undergo differentiation into macrophages [19,20]. A stock solution of PMA at 40 mg/ml in dimethyl sulfoxide (DMSO, SigmaAldrich, Saint Louis, MO) was diluted in tissue culture medium with the final DMSO concentration of 0.1 . Addition of 0.1 DMSO alone did not cause THP-1 cells to undergo macrophage differentiation, nor did it affect their viability as assessed using the MTT assay (data not shown).ConclusionsIn response to societal pressures to refine, reduce and replace the use of animals in experimentation, the increasing costs associated with animal models, and the advances in bioinformatics and systems biology, in vitro model systems are an increasingly important tool in biomedical science. While there are limitations associated with cell lines, particularly those that have been immortalized and thus express significant mutations that may alter the physiology of these cells relative to the primary cell type from which they were derived, cell lines, particularly those of human origin such as the THP-1 cell line, are especially useful for pilot projects, drug and toxicity screening, biochemical studies of signal transduction pathways and other types of studies that require large number of cells. Although widely used, standard tissue culture methods expose cells to oxygen levels considerably higher than those encountered by most cells under physiological conditions, and our data corroborate earlier studies in other cell types suggesting that altering oxygen tension impacts cell behavior. Regulating oxygen levels to optimize cell function in vitro is notProliferation AssaysNon-differentiated THP-1 cells were synchronized by serum deprivation for 48 h prior to being plated at an initial density of 0.76106 cells/ml in 35 mm tissue culture dishes and cultured under the conditions indicated in Figure 1. At 24 or 48 h after plating, cell density was determined using a hemocytometer. The percent growth was calculated according the following equation: [(final cell density at 24 or 48 h *100)/(0.76106)] – 100). Experiments were independently repeated five times with 3 samples per treatment in each experiment.Oxygen Tension Influences THP-1 Cell PhysiologyMetabolic Activity AssaysThe metabolic activity of the cells was evaluated by.Actor NF-kB results in the secretion of embryonic alkaline phosphatase (SEAP), which is detected 23388095 using Quanti-Blue reagent (Invitrogen). Cells were maintained in RPMI 1640 medium containing 11.11 mM glucose in but no phenol red (GIBCO, Carlsbad, CA) supplemented with 10 heat-inactivated fetal bovine serum (FBS, GIBCO), 1 penicillin (GIBCO), 1 streptomycin (GIBCO) and 50 mM 2-ME (Fisher Scientific, Pittsburgh, PA) at 37uC in a humidified incubator with 5 CO2 and 95 air (e.g., standard culture conditions). Prior to experimentation, the cells were starved for 48 h and subsequently cultured with or without 2-ME and/or FBS at 37uC in a humidified Thermo Scientific CO2 tissue culture incubator (NAPCO Series 8000WJ, Thermo Forma, Marietta, OH) equipped with built-in CO2 and O2 monitors and attached nitrogen and carbon dioxide gas supplies. Carbon dioxide was set to 5 v/v and oxygen to 5 of 18 . The oxygen and carbon dioxide contents of the incubator atmosphere were periodically verified using a Fyrite gas analyzer (Bacharach Inc., New Kensington, PA). For some experiments, cultures were treated for 24 or 48 h with phorbol 12-myristate 13-acetate (PMA, SigmaAldrich, St. Louis, MO) at 20 ng/ml to trigger 16985061 THP-1 cells to undergo differentiation into macrophages [19,20]. A stock solution of PMA at 40 mg/ml in dimethyl sulfoxide (DMSO, SigmaAldrich, Saint Louis, MO) was diluted in tissue culture medium with the final DMSO concentration of 0.1 . Addition of 0.1 DMSO alone did not cause THP-1 cells to undergo macrophage differentiation, nor did it affect their viability as assessed using the MTT assay (data not shown).ConclusionsIn response to societal pressures to refine, reduce and replace the use of animals in experimentation, the increasing costs associated with animal models, and the advances in bioinformatics and systems biology, in vitro model systems are an increasingly important tool in biomedical science. While there are limitations associated with cell lines, particularly those that have been immortalized and thus express significant mutations that may alter the physiology of these cells relative to the primary cell type from which they were derived, cell lines, particularly those of human origin such as the THP-1 cell line, are especially useful for pilot projects, drug and toxicity screening, biochemical studies of signal transduction pathways and other types of studies that require large number of cells. Although widely used, standard tissue culture methods expose cells to oxygen levels considerably higher than those encountered by most cells under physiological conditions, and our data corroborate earlier studies in other cell types suggesting that altering oxygen tension impacts cell behavior. Regulating oxygen levels to optimize cell function in vitro is notProliferation AssaysNon-differentiated THP-1 cells were synchronized by serum deprivation for 48 h prior to being plated at an initial density of 0.76106 cells/ml in 35 mm tissue culture dishes and cultured under the conditions indicated in Figure 1. At 24 or 48 h after plating, cell density was determined using a hemocytometer. The percent growth was calculated according the following equation: [(final cell density at 24 or 48 h *100)/(0.76106)] – 100). Experiments were independently repeated five times with 3 samples per treatment in each experiment.Oxygen Tension Influences THP-1 Cell PhysiologyMetabolic Activity AssaysThe metabolic activity of the cells was evaluated by.