Assays that use dyes such as trypan blue or propidium iodide are based on the concept that these dyes will be prevented from entering the cell unless there is disruption to the cells membrane (Strober, 2001). Hence healthy cells will remain unstained, while dead cells will stain positive. The amount of dye within a cell population can be measured and used to determine the percentage of cytotoxic cells. One limitation with this approach is that it only stains dead cells whilst dying or unhealthy cells may remain unstained. Alternatively a dye such as crystal violet can
stain deoxyribonucleic acid (DNA) within a cell as shown (Fig. 5). In this assay the color absorbance of the stained cells can be measured at a wavelength of approximately 570 nm, which can then check details be used to assess ABT-888 purchase the number of cells present (Gillies et al., 1986 and Rothman, 1986). A reduction in cell number would indicate a cytotoxic effect. In the neutral red assay, lysosomes rather than DNA in healthy cells are stained positive. The dye can then be extracted and used to quantify the number of viable cells (Repetto et al., 2008). Fotakis and Timbrell (2006) found
that the neutral red assay was more sensitive to cytotoxic effects on cells than several other assays tested. In addition to staining, DNA can be quantified using other techniques. For example in a thymidine incorporation assay, 3H-thymidine (a radioactive nucleoside) is incorporated into newly synthesized DNA during mitosis. Inhabitation of thymidine incorporation would indicate cytotoxicity. Protein Carnitine dehydrogenase assays have been used to determine cytotoxicity by measuring protein content within cells. A reduction
in protein concentration would correspond to a decrease in the number of cells. Coomassie brilliant blue protein assays (also referred to as the Bradford assay) is a colorimetric protein assay that can be used to quantify cellular protein by measuring the color absorbance from stained cells. Similarly, the Lowry test measures the amount of cellular protein by reacting copper ions to amino acids in proteins under alkaline conditions and measuring a subsequent color change. Enzymatic assays are among the most commonly used to assess cytotoxicity. LDH assays quantify the release of LDH following rupture of the cell membrane by using it to catalyze the conversion of lactate to pyruvate which can be measured colormetrically and used to quantify cell death. MTT assays measures the reduction of yellow MTT to purple formazan by mitochondrial succinate dehydrogenase. This change in color is measurable via spectrophotometry. As MTT reduction only occurs in metabolically active cells, the spectrophotometer reading can give an estimate of the number of viable cells present. The short time exposure test (STE) is a relatively simple assay method that estimates cell cytotoxicity and viability using MTT (Kojima et al., 2013, Takahashi et al., 2008 and Takahashi et al., 2011).