Cell-viability assay

Typical Phenotypic Assays 10.43.1 Cell-Viability Assay 

Enzymatic activity within hve cells is an alternative option for measuring cell viabiUty. A cell-permeable fluorogenic protease substrate (GF-AFC) has recendy been developed to selectively detect protease activity that is restricted to viable cells [80]. After GF-AFC penetrates into liver cells, the cytoplasmic aminopepti-dase cleaves the substrate by removing amino acid residues Gly and Phe. The final product aminofluorocoumarin (AFC), is released, which generates a fluorescent signal proportional to the quantity of viable cells. ATP has been widely 

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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. 

MTS cytotoxicity data were collected for a comparison of (R,/ )-psorospermin with (R,R)-5-methoxy-psorospermin, (.R,S()-5-methoxy-psorospermin, (5, / )-5-methoxy-psorospermin, and (N,S()-5-methoxy-psorospermin in cell viability assays with various tumor cell lines. Test results are provided in Table 1. 

Table 1 IC50 MTS cytotoxicity data with experimental agents in cell viability assays with various tumor cell lines...

Fig. 17.15 The cell viability assay to explore the function of small molecules as promoters of apoptosis.

CellTiter-Blue cell viability assay, the mean % cell vitality growth for the fractions of each of the five plant species C. arborea, C. erythrocarpos, D. steudneri, L. schimperi and S. ellipticum were 43.1%, 67.2%, 82.1%, 52.3% and 87.6% respectively, with PO.OOOl and 95% confidence intervals (Cl) of 54.746-81.082 pg/ml. The IC50 concentration for compound Lup-20(29)-en-3-one (1) was 1.747x10-6 pM with 95% confidence intervals (Cl) of 3.019xl0 7 to 1.01 lxlO"4 pM. Results indicate that most of the extracts tested were relatively non-toxic hence supporting the inherent use of these plants in traditional medicine. 

Brine shrimp lethality and cell line culture assays are the most frequently used in estimating preliminary toxicity potential of different biological agents. In this study, brine shrimp and CellTiter-Blue Cell Viability Assays were used in evaluating the cytotoxity potential of different plant extracts. 

Cell viability assay (MTS assay) was performed by incubating STPP liposomes with 4T1 cells grown in 96-well plates. Data obtained from this assay was used to establish the amount of STPP liposomes that can be safely used with cells (3). 

Cell lines have been used extensively to study the cytotoxicity of substances to fish cells12,184. Most studies have employed tests of general or basal cytotoxicity rather than tests of injury to differentiated cells and their functions. Basal cytotoxicity refers to impairment to cellular activities shared by all or most cells. Evaluating basal cytotoxicity can be done in a variety of ways, which will be referred to as cell viability assays. Usually these tests are performed on cultures after exposure to putative toxicants for 72 h or less and can be described as short-term or acute assays. As a result of this short exposure, toxicants that act by inducing a particular cellular process, such as the xenobiotic metabolism, or by causing cumulative damage might be missed. 

Cell viability assays can be profoundly influenced by the exposure medium. Usually less cytotoxicity is observed when the basal medium is supplemented with serum, such as FBS. This is true not only for heavy metals but also for organic ecotoxicants184. Although the basis of this protective action likely depends to a degree on the specific toxicant, three general mechanisms to be considered are toxicant availability, toxicant uptake and the protective molecules of serum. For fluoranthene, FBS altered its distribution within the cell cultures and its availability to the cells175. 

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