Thymidine incorporation is highly predictive of colony formation and can be used for high-throughput screening

The primary goal of anticancer chemotherapy is to kill cancer cells. Therefore, it is of critical importance that any assay that is used to determine the toxicity of a potential anticancer drug accurately measures viability. While colony formation is widely regarded as the most accurate measure of viability following drug treatment, it is laborious, time consuming, and difficult to carry out with non-adherent cells. For these reasons, it is not suitable for moderate- to high-throughput screening applications. We sought to identify a convenient and reliable assay that would accurately reproduce colony formation results and be amenable to high-throughput applications. Here, we describe a modification of the 3H-thymidine incorporation assay that meets these criteria. The assay can be carried out in 96-well plates with minimal handling of reagents and media. It can be performed with non-adherent and adherent cell lines. Most importantly, LC50 values obtained with this assay show excellent agreement with colony formation results. Taken together, these advantages make the modified 3H-thymidine incorporation assay well suited for high-throughput viability assays in anticancer drug discovery and development.     

The fluorometric microculture cytotoxicity assay

The fluorometric microculture cytotoxicity assay (FMCA) is a nonclonogenic microplate-based cell viability assay used for measurement of the cytotoxic and/or cytostatic effect of different compounds in vitro. The assay is based on hydrolysis of the probe, fluorescein diacetate (FDA) by esterases in cells with intact plasma membranes. The assay is available as both a semiautomated 96-well plate setup and a 384-well plate version fully adaptable to robotics. Experimental plates are prepared with a small amount of drug solution and can be stored frozen. Cells are seeded on the plates and cell viability is evaluated after 72 h. The protocol described here is applicable both for cell lines and freshly prepared tumor cells from patients and is suitable both for screening in drug development and as a basis for a predictive test for individualization of anticancer drug therapy.     

Defect in cell wall integrity of the yeast saccharomyces cerevisiae caused by a mutation of the GDP-mannose pyrophosphorylase gene VIG9

The Saccharomyces cerevisiae VIG9 gene encodes GDP-mannose pyrophosphorylase, which synthesizes GDP-mannose from GTP and mannose-1-phosphate. Although the null mutant was lethal, the vig9 mutants so far obtained showed no growth defect but immature protein glycosylation and drug hypersensitivity. During our search for cell-wall mutants, we found a novel temperature-sensitive mutant, JS30, which required an osmotic stabilizer for viability. JS30 excreted cell surface proteins in the medium without any indication of cell lysis. Although conventional genetic analysis using mating was impossible, by detailed characterization of JS30 including an in vitro enzyme assay and nucleotide sequencing, we found the defect of JS30 was due to a mutation in the VIG9 gene. These results indicated a critical role of GDP-mannose in maintenance of cell-wall integrity.     

Use of the tetrazolium salt MTT to measure cell viability effects of the bacterial antagonist Lysobacter enzymogenes on the filamentous fungus Cryphonectria parasitica

Despite substantial interest investigating bacterial mechanisms of fungal growth inhibition, there are few methods available that quantify fungal cell death during direct interactions with bacteria. Here we describe an in vitro cell suspension assay using the tetrazolium salt MTT as a viability stain to assess direct effects of the bacterial antagonist Lysobacter enzymogenes on hyphal cells of the filamentous fungus Cryphonectria parasitica. The effects of bacterial cell density, fungal age and the physiological state of fungal mycelia on fungal cell viability were evaluated. As expected, increased bacterial cell density correlated with reduced fungal cell viability over time. Bacterial effects on fungal cell viability were influenced by both age and physiological state of the fungal mycelium. Cells obtained from 1-week-old mycelia lost viability faster compared with those from 2-week-old mycelia. Likewise, hyphal cells obtained from the lower layer of the mycelial pellicle lost viability more quickly compared with cells from the upper layer of the mycelial pellicle. Fungal cell viability was compared between interactions with L. enzymogenes wildtype strain C3 and a mutant strain, DCA, which was previously demonstrated to lack in vitro antifungal activity. Addition of antibiotics eliminated contributions to MTT-formazan production by bacterial cells, but not by fungal cells, demonstrating that mutant strain DCA had lost complete capacity to reduce fungal cell viability. These results indicate this cell suspension assay can be used to quantify bacterial effects on fungal cells, thus providing a reliable method to differentiate strains during bacterial/fungal interactions.     

Assessment of the antitumour activity of targeted immunospecific albumin microspheres loaded with cisplatin and 5-fluorouracil: toxicity against a rodent ovarian carcinoma in vitro

The 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay is used successfully to estimate the number of viable cells in drug screening trials. We used the MTT assay to assess the viability of a rodent ovarian carcinoma cell line (DMBA-OC-1R) after exposure to combinations of cisplatin and 5-fluorouracil as free drug and in encapsulated (conjugated and unconjugated) forms. After 48 h of exposure to free drugs, a significant trend towards cell cytotoxicity could be observed and this was well established by 120 h. Cells treated with drug-containing immuno-microspheres showed a similar initial decrease in cell viability after 96 h, and this was maintained for 128 h. These results suggest that immuno-microspheres loaded with chemotherapeutic drugs have the potential to be successfully used in the treatment of ovarian cancer.     

Propidium iodide-based methods for monitoring drug action in the kinetoplastidae: comparison with the Alamar Blue assay

The urgent need for new drug development for African trypanosomiasis is widely recognized. This requires reliable and informative high-throughput assays. Currently, drug action is determined with a fluorimetric/colorimetric assay based on the metabolism of the dye Alamar Blue (resazurin) by live cells. However, this assay does not easily distinguish between cell death and growth arrest, or supply information about the rate at which test compounds affect these parameters. We report here an alternative fluorimetric assay, based on the interaction of propidium iodide with DNA, that allows either real-time monitoring of cell viability or the generation of EC₅₀ values at a predetermined time-point. The assay is highly sensitive and fluorescence readings easily correlate to numbers of parasites or DNA content. The EC₅₀ values were highly similar to those obtained with the standard Alamar Blue assay. The procedure lends itself readily to applications in drug development or resistance monitoring.     

A flow-cytometric method for determination of yeast viability and cell number in a brewery

A flow-cytometric assay, using the fluorescent dye, oxonol, for the simultaneous determination of yeast cell viability and cell number is described. The assay was optimised, and trialed at a brewery for 6 months. The flow-cytometry assay offered a substantially reduced error in viability determination, compared to methylene blue which is the industry standard for measuring viability. Further, by calculating yeast cell number at the same time, this assay provides a reliable method for determining pitching rate, allowing increased quality control of subsequent fermentations.     

Impact of combined mTOR and MEK inhibition in uveal melanoma is driven by tumor genotype

Uveal melanomas possess activation of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT/mammalian Target of Rapamycin (mTOR) pathways. MAPK activation occurs via somatic mutations in the heterotrimeric G protein subunits GNAQ and GNA11 for over 70% of tumors and less frequently via V600E BRAF mutations. In this report, we describe the impact of dual pathway inhibition upon uveal melanoma cell lines with the MEK inhibitor selumetinib (AZD6244/ARRY-142886) and the ATP-competitive mTOR kinase inhibitor AZD8055. While synergistic reductions in cell viability were observed with AZD8055/selumetinib in both BRAF and GNAQ mutant cell lines, apoptosis was preferentially induced in BRAF mutant cells only. In vitro apoptosis assay results were predictive of in vivo drug efficacy as tumor regressions were observed only in a BRAF mutant xenograft model, but not GNAQ mutant model. We went on to discover that GNAQ promotes relative resistance to AZD8055/selumetinib-induced apoptosis in GNAQ mutant cells. For BRAF mutant cells, both AKT and 4E-BP1 phosphorylation were modulated by the combination; however, decreasing AKT phosphorylation alone was not sufficient and decreasing 4E-BP1 phosphorylation was not required for apoptosis. Instead, cooperative mTOR complex 2 (mTORC2) and MEK inhibition resulting in downregulation of the pro-survival protein MCL-1 was found to be critical for combination-induced apoptosis. These results suggest that the clinical efficacy of combined MEK and mTOR kinase inhibition will be determined by tumor genotype, and that BRAF mutant malignancies will be particularly susceptible to this strategy.     

Cell-based chip for the detection of anticancer effect on HeLa cells using cyclic voltammetry

HeLa cells directly immobilized on gold-patterned silicon substrate were used to assess the biological toxicity of anticancer drugs (hydroxyurea and cyclophosphamide). Immobilization of HeLa cells was confirmed by optical microscopy, and cell growth, viability and drug-related toxicity were examined by cyclic voltammetry and potentiometric stripping analysis. The voltammetric behaviors of HeLa cells displayed a quasi-reversible pattern with the peak current exhibiting a linear relationship with cell number. The attached living cells were exposed to different concentrations of hydroxyurea and cyclophosphamide as anticancer drugs, which induced the change of cyclic voltammetry current peak. As the exposed concentration of anticancer drugs was increased, the change of current peak was increased, which indicates the decrease of cell viability. Trypan Blue dyeing was performed to confirm the results of the effect of anticancer drugs on the cell viability which was obtained from cyclic voltammetry assay. The proposed direct cell immobilization method technique can be applied to the fabrication of cell chip for diagnosis, drug detection, and on-site monitoring.     

Decrease in cell viability in an RMF,sigma(38), and OmpC triple mutant of Escherichia coli

In a speG-disrupted Escherichia coli mutant, which cannot metabolize spermidine to acetylspermidine, addition of spermidine to the medium caused a decrease in cell viability at the late stationary phase of growth. There were parallel decreases in the levels of ribosome modulation factor (RMF), the sigma(38) subunit of RNA polymerase, and the outer membrane protein C (OmpC). To clarify that these three proteins are strongly involved in cell viability, the rmf, rpoS (encoding sigma(38)), and ompC genes were disrupted. Viability of the triple mutant decreased to less than 1% of normal cells. The triple mutant had a reduced cell viability compared to any combination of double mutants, which also had a reduced cell viability. The single rmf and rpoS, but not ompC, mutant only slightly reduced cell viability. The results indicate that cooperative functions of these three proteins are necessary for cell viability at the late stationary phase. The triple mutant had a reduced level of ribosomes and of intracellular cations.     

The effect of the bisphosphonate alendronate on viability of canine osteosarcoma cells in vitro

The objective of this study was to determine the effect of alendronate on the viability of canine osteosarcoma cells and nonneoplastic canine cells. The sample population was composed of canine osteosarcoma tumor cells. Osteosarcoma cells and canine fibroblasts were maintained in culture under standard conditions. The MTT assay for cell viability was performed after 24, 48, and 72 h of incubation with alendronate (0.001 to 1000 microM) or no drug (control). Plates were set up so that each concentration and the control had a sample number of 8. The optical density (OD) of each well was measured at 540 nm using an enzyme-linked immunosorbent assay microplate reader. The percent viability was determined for each concentration and for each incubation time. After 24 h of incubation of POS (parent osteosarcoma) and HMPOS cells with alendronate, there was no significant difference in mean OD at any drug concentration when compared with control samples. A significant concentration- and time-dependent reduction in mean OD of osteosarcoma cells was observed after 48 and 72 h of incubation, with alendronate concentrations ranging from 10 to 1000 microM. The lowest percent cell viability observed in treated cells was 35%. Conversely, alendronate did not significantly affect mean OD in fibroblasts, and the lowest percent cell viability observed was 76%. Our data indicate that alendronate may have the potential to inhibit canine osteosarcoma tumor growth. It will be important to determine the clinical relevance of these in vitro findings. If similar findings are observed in vivo, use of alendronate may also be indicated as an adjuvant to existing chemotherapeutic protocols.     

Fluorescence-based viability assay for studies of reactive drug intermediates

Studies of drug toxicity, toxicologic structure-function relationships, screening of idiosyncratic drug reactions, and a variety of cytotoxic events and cellular functions in immunology and cell biology require the sensitive and rapid processing of often large numbers of cell samples. This report describes the development of a high-sensitivity, high-throughput viability assay based on (a) the carboxyfluorescein derivative 2'-7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) as a vital dye, (b) instrumentation capable of processing multiple small (less than 100 cells) samples, and (c) a 96-well unidirectional vacuum filtration plate. Double staining of cultured peripheral blood mononuclear cells with BCECF and propidium iodide (PI) showed no overlap between PI+ (nonviable) and BCECF+ (viable) cells by flow cytometric analysis. Optimal conditions were developed for dye loading and minimizing physical cell damage and fluorescence quench during the assay procedure. The ratio of BCECF fluorescence to internal standard fluorescent particles was linear from 40 to greater than 20,000 cells with a signal:noise ratio of approximately 3 at 40 cells/well. Sulfamethoxazole hydroxylamine (SMX-HA) was used as a model toxic drug metabolite to explore the validity of the BCECF procedure. SMX-HA, but not its parent compound sulfamethoxazole, resulted in a dose dependent loss of cellular fluorescence and the parallel accumulation of PI+ nonviable cells. When compared to the currently used tetrazolium dye reduction viability assay, the BCECF method was 3-fold more sensitive, greater than 10-fold faster, and required 1/10-1/100 the cell numbers.     

A high throughput screen for inhibitors of fungal cell wall synthesis

Fungal cell wall synthesis is essential for viability, requiring the activity of genes involved in environmental sensing, precursor synthesis, transport, secretion, and assembly. This multitude of potential targets, the availability of known agents targeting this pathway, and the unique nature of fungal cell wall synthesis make this pathway an appealing target for drug discovery. Here we describe the adaptation of an assay monitoring cell wall synthesis for high-throughput screening. The assay requires fungal cell growth, in the presence of the test compound, for 3 h before the cells are subjected to osmotic shock in the presence of a dye that stains DNA. Miniaturization of the assay to a 384-well plate format and removing a mechanical transfer led to subtle changes in the assay characteristics. Validation of the assay with a library of known pharmacologically active agents has identified a number of different classes of compounds that are active in this assay, causing aberrant cell wall morphology and in many cases the inhibition of fungal cell growth.     

Comparative study with the alkaline Comet assay and the chromosome aberration test

The alkaline Comet assay is becoming a useful tool for early genotoxicity testing of new pharmaceutical drug candidates. The aim of this study was to elucidate the predictive value of Comet assay results for the outcome of the chromosome aberration (CA) test. For this purpose, a validation exercise with 13 drug candidates was carried out utilizing V79 Chinese hamster cells and human lymphocytes. The study demonstrates that results of the Comet assay and the chromosome aberration test show a high degree of agreement, irrespective of the cell type used. In the Comet assay, seven compounds were positive and six were negative, while in the CA test, six were positive and seven were negative. The only discrepancy was found with one compound that was positive in the Comet assay with V79 cells, negative in the Comet assay with human lymphocytes and clearly negative in the CA test with human lymphocytes. For the selection of concentrations for testing in the Comet assay, cytotoxicity by means of cell count after incubation or viability by means of Trypan-blue dye exclusion (TBDE) were used. The results show that either parameter led to analysis of a concentration range in the Comet assay similar to that chosen in the CA test, in which cell count (when using V79 cells) or mitotic index (in case of lymphocytes) were used. However, since cell count after incubation of cells is much more labour-intensive, viability was preferred as the parameter to assess cytotoxicity and for selecting concentrations for analysis in the Comet assay. The data presented in this study may contribute the regulatory acceptance of the Comet assay, e.g. for mechanistic studies.     

A versatile assay for the accurate,time-resolved determination of cellular viability

A convenient and versatile method for the accurate, time-resolved determination of cellular viability has been developed. The conventional viability indicator fluorescein diacetate (FDA), which is converted to the fluorescent compound fluorescein in living cells, was employed as a viability probe. Fluorescence emission from cells was measured using a spectrofluorimeter equipped with a magnetic stirrer. Using this assay cell suspensions exhibiting densities in the range 0.5 x 10(5) to 2.0 x 10(5) cells displayed a linear response when FDA concentrations less than 12 micro M were employed. To calibrate the method, viability standards were elaborated using different proportions of living and dead cells, and a correlation coefficient for the viability of tobacco BY-2 suspensions was calculated as 0.998. This viability assay was also found to be applicable to Chlamydomonas reinhardtii and Arabidopsis thaliana cultured cells. Using this cell viability assay, kinetic analyses of cell death could be performed. Using the proteinaceous elicitor from Phytophthora cryptogea, cryptogein, to induce cell death in tobacco cell suspensions, values for the maximum velocity of death induction rate (V(max)) and the LD50 (half-maximal velocity or k(1/2)) were calculated as 17.2 (% death/h) and 65 nM, respectively.     

Overlapping functions for recF and priA in cell viability and UV-inducible SOS expression are distinguished by dnaC809 in Escherichia coli K-12

The recF and priA genes have roles in DNA repair and homologous recombination. Mutations in these genes also cause decreases in cell viability and alterations in UV-inducible sulAp–lacZ (SOS) expression. To find out if the two genes are in the same or different pathways for viability and SOS expression, the phenotypes of the double mutant strains were studied. The recF priA double mutant showed a lower viability and SOS expression level than either of the single mutants. In the case of cell viability, recF missense mutations decreased viability of a priA2 :: kan strain two to fivefold whereas recF null priA2 :: kan double mutants were not viable at all. dnaC809 , a mutation that suppresses the UV-sensitive (UV^S) and Rec⁻ phenotypes of priA2 :: kan , restored cell viability, but not UV-inducible SOS expression, to a priA recF strain. Since recF is epistatic with recO and recR ( recOR ) for UV resistance, recOR mutations were also tested with priA2 :: kan . No overlap was found between recOR and priA for viability and SOS expression. It is concluded that priA and recF have two different overlapping functions in viability and SOS expression that are distinguishable by the effects of dnaC809 . The role of recF in a priA2 :: kan strain in cell viability is a new function for recF and unlike recF  's other roles in DNA repair and recombination, is independent of recOR . A new role for priA in UV-inducible SOS expression in a recF mutant is also defined.     

The determination of cellular viability of hybridoma cells in microtitre plates: A colorimetric assay based on neutral red

A colorimetric assay utilising Neutral Red (C.I. 50040), a nuclear stain, was developed to determine the cellular viability of hybridoma cells in microtitre plates. A linear correlation (r=0.99) was found to exist between the uptake of Neutral Red by viable cells and the viable cell count determined by Trypan blue exclusion test. The linearity stretched over the range of cell concentrations normal in batch cultures (2–30×10⁴/0.2 ml) with as little as ±6% intra-plate well-to-well variation and ±10.2% inter-assay variation.Microscopical examinations of viable hybridoma cells stained with Neutral Red showed that it was located in the nucleus. The possble bifunctional activity of the Neutral Red assay as a test for cellular viability and estimating the DNA content of hybridoma cells is discussed along with its application in a drug screening programme.     

Transformation Products of Carbamazepine (CBZ) After Ozonation and their Toxicity Evaluation Using <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. Strain KSH-1 in Aqueous Matrices

Carbamazepine (CBZ) is an anti-epileptic and anti-convulsant drug widely used for the treatment of epilepsy and other bipolar disorders. Ozone as an advanced oxidation process has been widely used for the degradation of CBZ resulting in the formation of transformation products (ozonides). The present research aims to isolate and identify potential microorganism, capable of degradation of CBZ and its transformation products. The cell viability and cytotoxicity of pure CBZ and their ozone transformation products were evaluated using the cells of Pseudomonas sp. strain KSH-1 through cell viability assay tests. The cells metabolic activity was assessed at varying CBZ concentrations (~?10–25 ppm, pure CBZ) and cumulatively for ozone transformation products. For pure CBZ, % cell viability decreases as CBZ concentration increases, while, in case of post-ozonated CBZ transformation products, the viability decreases initially and then increases upon exposure of ozone with a maximum cell viability of 97?±?2.8% evaluated for 2 h post-ozonated samples.     

Average cell viability levels of human dental pulp stem cells: an accurate combinatorial index for quality control in tissue engineering

Background aimsOne of the most important issues in tissue engineering (TE) is the search for a suitable stem cell reservoir with optimal cell viability levels for the development of new tissues relevant for therapeutic needs. The aim of this study was to evaluate the cell viability levels of 10 sequential cell passages of human dental pulp stem cells (hDPSC) to determine their potential for TE techniques.MethodsTo assess the average cell viability levels of hDPSC, four cell viability assays were used in a combinatorial approach: trypan blue exclusion test, water-soluble tetrazolium 1 assay, live/dead assay and electron probe x-ray microanalysis.ResultsThe results showed that cell viability as determined by trypan blue staining and live/dead assays was greater than 85%, with a significant decrease at the second passage (P < 0.05) and a significant increase at the ninth passage (P < 0.05). Electron probe x-ray microanalysis showed that the highest cell viability corresponded to the ninth passage, with the lowest K/Na values found at the third passage. No statistical differences were found among the different passages for the water-soluble tetrazolium 1 assay (P = 0.219).ConclusionsAssessment of average cell viability levels showed that the highest viability of hDPSC was reached after nine passages, suggesting that this passage would be the most adequate for use in TE protocols.