Viability assays

At first glance, the in vitro studies on CNT toxicity appear to be confusing, inconclusive, or contradictory. However, if one considers interference with dye-based viability assays, agglomeration issues, and oxidative stress due to catalyst contamination, the data available to date seem to favor the conclusion that well-dispersed, purified, and/or functionalized CNTs exhibit relatively low toxicity. 

Worle-Knirsch, J.M., Pulskamp, K. and Krug, H.F. (2006) Oops they did it again Carbon nanotubes hoax scientists in viability assays. Nano Letters, 6 (6), 1261-1268. 

It should be kept in mind that high lipid concentrations and/or long incubation times may be toxic for cells and mimic results seen with reduced uptake. Therefore, all experiments need to be accompanied by viability assays, for... 

CellTiter-Glo Luminescent Gell Viability Assay (G7572, Promega). 

For viability assay, 96 h after the transfection, remove growth media by flicking the plate sideways above a sink and add 100 p.1 of DMEMrCell Titer Glo 1 1 solution. 

The use of trypsin is common to detach cells from adherent cultures however, in this case it is not recommended. MCF-7 cells treated with trypsin tend to form clumps, which are difficult to breakup. The use of Accutase yields a mostly single-cell suspension that will not form clumps even after it has been washed with labeling buffer. Total cell count and viability assays will also be easier for the researcher to carry out. 

FIGURE 8.10 Chronological sequence of images of a viability assay on a single Jurkat T cell, (a) Live cell perfused with trypan blue dye. Since the cell is alive, it is not stained, (b) Cell perfused with methanol, which causes cell membrane permeabilization, followed by cell death, (c) Permeabilized cell perfused with dye it is rapidly (< 5 s) stained. The entire assay is performed in less than 2 min [368]. Reprinted with permission from the American Chemical Society. 

Fig. 5 Anticancer activities of the isostructural kinase inhibitors DWI2 (M = Ru) and DWHOs (M = Os) against the 1205Lu melanoma cell line. LC50 curves were determined using the MTT cell viability assay after treatment with the inhibitors for 72 h. An average of five independent experiments is shown...

The following example describes the results of the dereplication of an HTP fraction library for inhibition of melanin formation in an in vitro assay with a B16 cell line [54]. Briefly, following the inhibition and cell viability assay, the active organic extract from the whole plant of Mallotus repandus was fractionated with HTP. All of the HTP fractions were tested for melanin inhibitory activities. As shown in Figure 8, there are three major peaks exhibiting > 50% inhibitions of melanin synthesis and seven minor peaks exhibiting weaker inhibitions. The sharp activity peaks indicate the quality of the separations, which distributed the active components in three to five cells. 

Since the melanin formation assay was run against a cell viability assay, the activity peak maximum at fraction Dll was most likely due to cytotoxicity. The dereplication of another active peak located from fractions D7 to D2 is illustrated in Figure 9. Every active fraction was analyzed by HPLC/PDA/MS. There was a peak located at Rt=16.33 min in the LC/MS total ion chromatogram of active fractions. This peak showed the same pattern of increasing to decreasing intensity as the trend exhibited by the melanin inhibition activities of those fractions. 

Among the first cell viability assays developed for HTS was the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] tetrazolium reduction assay (Mosmann 1983) that served as a milestone for this type of study. The assay offered a non-radioactive alternative to tritiated thymidine incorporation into DNA as a method of measuring cell proliferation. In many cases, the MTT assay can directly substitute for the tritiated thymidine incorporation assay (Figure 6.3). The MTT tetrazolium compound is prepared in a physiologically balanced solution, added to cells in culture, and incubated for approximately 4 hr. Viable cells convert MTT into an intensely colored formazan product that can be quantitated by recording changes in absorbance at specific wavelengths. 

Hanna et al. 2001 CellTiter-GlO luminescent cell viability assay a sensitive and rapid method for determining cell viability. Promega Cell Notes 2, 11-13. 

Tominaga, H. et al. 1999. A water-soluble tetrazolium salt useful for colorimetric cell viability assay. Anal. Commun. 36, 47-50. 

Both a short-term intracellular drug accumulation and a long-term viability assay have been used to understand the anticancer properties of the effects of flavonoids against tumor cells [14,16]. 

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