Model tc 20 cell counter

J774, a murine macrophage cell line, and Caco-2, a human intestinal epithelial cell line, were utilized to determine if chelation with nutraceuticals can reduce or eliminate the cytotoxicity associated with those metals considered likely internal contamination threats. Both cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). All cell culture reagents were purchased from Invitrogen (Grand Island, NY, USA). J774 cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum [19 (link)] while Caco-2 cells were cultured in F12-K medium supplemented with 20% fetal bovine serum [20 (link)]. Stock cultures were maintained in tissue culture flasks (75 cm² area) in a humidified atmosphere of 5% CO₂/95% air. For both cell lines, the medium was replaced every 2 to 3 days and cells were subcultured when 70–80% confluent based on direct microscopic observation. Cell numbers and viability assessments were conducted using trypan blue (Gibco) dye exclusion with the Bio-Rad Model TC-20 Cell Counter (Hercules, CA, USA).

Stock cell cultures were maintained in tissue culture flasks (75 cm² area) at 37 °C in a humidified atmosphere of 5% CO₂. For endothelial cells, the flasks were treated with 6 mL of Quick-Coat (Angio-Proteomie, Boston, MA, USA) to provide a suitable substrate for attachment. For both cell lines, medium was refreshed every other day and cells were subcultured when 70–80% confluent based upon direct microscopic observation. Briefly, medium was aspirated from the flasks and the flasks were washed once with phosphate-buffered saline (PBS, Gibco). After aspirating the PBS, 3 mL of StemPro Accutase Cell Dissociation Reagent (Gibco) were added and the flask left at room temperature for up to 8 min to allow the cells to dissociate from the flask. Once detached, fresh medium was added, and the cell suspension pipetted to break up any clumps. The cell suspension was centrifuged at 240× g for 10 min, after which the cell pellet was resuspended in a suitable amount of the appropriate medium and transferred to new flasks. Cell numbers and viability were determined using trypan blue (Gibco) exclusion with the Bio-Rad Model TC-20 Cell Counter (Hercules, CA, USA).