A colorimetric assay for the assessment of cytotoxicity of yeasts

A colorimetric assay for the quantitation of microbial cytotoxicity has been developed using cells from a monocyte-like human cell line (U937), epithelial cells (Hela), and fibroblast-like cells (Vero) as targets. The fraction of surviving cells was determined by their content of the dye neutral red which is retained only by live cells and can be quantitated photometrically after controlled lysis. The neutral red retention assay was at least as sensitive as the 51Cr-release assay; it was considerably less laborious, faster, and avoided handling of radioactivity. Among the different Candida species tested, the highest cytotoxicity was associated with C. albicans and C. tropicalis; a lower degree of cytotoxicity was exhibited by C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis, and C. pseudotropicalis. Among the strains of a given fungal species cytotoxicity varied by up to 40%.     

Comparison of the Neutral Red and Methylene Blue Assays to Study Cell Growth in Culture

The neutral red and methylene blue in vitro cytotoxicity assays were compared under a variety of conditions using normal human ovarian epithelial cells to determine whether either assay is superior for studying cell growth. The results were standardized against a DNA spectrofluorometric assay. Although the assays were equivalent in reflecting cell number, each has specific advantages: while neutral red discriminates between viable and dead cells, the methylene blue assay is more sensitive and easier to perform.     

Neutral red uptake and clonogenic survival assays of the hyperthermic sensitization of tumor cells to tumor necrosis factor

Vital dye uptake and postfixation dye assays have recently been used to examine the interaction between short-term (24-48 h) exposures to the monokine, tumor necrosis factor (TNF), and hyperthermic treatments with the finding that synergistic increases in cytotoxicity occurred. However, survival measured by these short-term dye assays is not necessarily closely related to eventual loss of clonogenic capacity. Treatment-induced growth delays, delayed cytotoxic effects, or perturbations of vital dye sequestration mechanisms could result in a different measurement of surviving fraction than given by a clonogenic assay. In this study we directly compared the neutral red vital dye uptake and clonogenic survival assays and confirmed in both assays that TNF-sensitive (L-929) and TNF-resistant (EMT-6) phenotypes show greatly reduced survival when treated with combined recombinant human TNF (1.0-0.0005 micrograms/ml) and hyperthermia (1-2 h at 43 degrees C). Moreover, we confirmed that sensitization of the TNF-resistant EMT-6 cells was largely dependent on monokine treatment before hyperthermia and was reduced by the reverse sequence. The greatest sensitization of TNF-responsive L-929 cells also occurred when TNF treatment preceded heating. These results for clonogenic survival are consistent with the hypothesis that hyperthermia used in combination with TNF in vivo is more cytotoxic than TNF or hyperthermia separately.     

Cross contamination associated with the use of multiwell culture plates for cytotoxicity assessment of volatile chemicals

In vitro toxicity testing can involve technical problems due to the evaporation of volatile test chemicals. The cytotoxicity of two volatile chemicals (butanol and ethanol has been assessed with neutral red assay in conventional microtiter plates. The non volatile DMSO chemical is used as a negative control. Under these conditions, an important cross contamination between test concentration groups has been observed. This affects cytotoxicity estimation which is overestimated. This cross contamination is prevented when special plates containing removalbe bars are used.     

Cell-mediated immunity to Friend virus-induced leukemia. I. Modification of 125IUdR release cytotoxicity assay for use with suspension target cells.

Cell-mediated cytotoxic reactivity of C57BL/6 mice against syngeneic FBL-3 cells, a Friend virus-induced leukemia, was measured by the 125IUdR release assay with tumor target cells in suspension. The tests could be performed either with Linbro tissue culture plates(16-mm wells) or with Microtest II plates (6-mm wells). The former could only be harvested manually; the latter could be harvested mechanically by an automatic harvesting apparatus which permitted larger scale tests. With either plate, it was found that careful preparation of the target cells and of the attacker cells has important effects on the results obtained. When performed under optimal test conditions, the 125IUdR assay can be used as a very simple, objective, and reproducible assay for testing cell-mediated cytotoxicity.     

An improved resazurin-based cytotoxicity assay for hepatic cells

A simple resazurin-based cytotoxicity assay is presented for screening of cytotoxicity in hepatocytes and liver cell lines. Human hepatoma (HepG2) cells in 96-well culture plates were exposed to known toxic (cisplatin, 5-fluorouracil, ethionine, flufenamic acid, and diflunisal) and control (transplatin, 5-chlorouracil, methionine, and acetylsalicylic acid) compounds for 1–3 days, and resazurin (5 μmol/L) was added. A conventional short-term (1 h) assay was first performed, where cytotoxicity is indicated by decreased reduction of resazurin to its fluorescent product resorufin. Our improved assay consists of additionally measuring fluorescence 2–4 days later, when cytotoxicity is indicated by a striking increase in the concentration of resorufin, resulting from two distinct processes. First, viable liver-derived cells slowly convert resorufin to nonfluorescent metabolites. Fluorescence of control cell wells decreased to background during a 2- to 4-day exposure to resazurin. This metabolism of resorufin was largely blocked by dicumarol and to lesser extents by disulfiram and SKF525a. Second, dead or dying cells slowly convert resazurin to resorufin but do not further metabolize resorufin; thus this fluorescent metabolite accumulates to high levels in wells with dead cells by 2 to 4 days. A similar increase in fluorescence associated with cytotoxicity was observed in primary cultures of rat hepatocytes using the long-term resazurin-based assay. In addition to an improved signal relative to the short-term assay, the inversion of the fluorescent signal from high = alive short-term to high = dead long-term allows determination of two independent cytotoxicity endpoints after addition of one innocuous vital dye. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of copper ions released from metallic copper on CHO-K1 cells

The aim of this study was to investigate the cytotoxic and genotoxic effect of copper extracts obtained from metallic copper in Chinese hamster ovary (CHO-K1) cell line using neutral red (NR), sister chromatid exchange (SCE), chromosomal aberrations (CA) and cell-cycle kinetics tests. Cells were cultured in Ham-F10 with different copper-containing extracts obtained after the immersion of copper disks for 1, 2, 3, 9, 12, 24, 48 and 72 h in culture medium. Results from cytotoxicity assay showed an inverted U-shape response evidenced in changes in lysosomal activity and mitotic index. The analysis of CA revealed an increase of abnormal metaphases for copper concentration (cCu) in the 5.67-7.42 mg/L dose-range (p<0.001). In addition, SCE frequencies were higher for treated cells when compared with controls in the 1.56-7.42 mg/L concentration range (p<0.001). The absence of metaphases indicated cytotoxicity for cCu≥10.85 mg/L. Results show that cells close to copper-containing materials releasing copper ions are susceptible to cytotoxic and genotoxic effects.     

Rapid detection of cytotoxicity of food additives and contaminants by a novel cytotoxicity test, menadione-catalyzed H2O2 production assay

Menadione-catalyzed H₂O₂ production by viable animal cells was proportional to the viable cell number, and H₂O₂ production decreased with increasing cytotoxic effects after the incubation of cells with cytotoxic compounds. The cytotoxic effects of food additives, pesticides, antibiotics, heavy metals, phytotoxins, mycotoxins, and marine toxins were estimated using the above test employingNIH/3T3 and Neuro-2a cells. Synergistic effects of the toxin mixture were observed and acute cytotoxicity detected 1 h after the incubation of cells with toxins. This menadione-catalyzed H₂O₂production assay is rapid and simple compared to other popular cytotoxicity tests such as the MTT reduction assay and Neutral red inclusion test, requiring4 h. The menadione-catalyzed H₂O₂ production assay is expected to be a useful food safety test for rapidly detecting toxic compounds having a basic cytotoxic effect on common animal cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Limitations of alginate gels as a culture model for the study of the effects of UVA radiation on human dermal fibroblasts

Several studies were undertaken to develop three-dimensional (3-D) cell culture models that allow conditions closer to the in vivo situation. To this end, alginate gels were tested as a 3-D cell culture model that might be useful in the study of the effects of UVA on human dermal fibroblasts. Cell culture in alginate gels and the irradiation conditions were optimized. Results showed that optimized cultures in alginate gels experienced considerable cell death on UVA irradiation compared to the classical monolayer cell culture. Viability tests (cell counting and neutral red assay) were performed to show that only UVA-irradiated alginate gels were responsible for this cytotoxicity. The implication of oxygen species in the phototoxicity induced by ultraviolet light has already been described; for this reason we investigated whether oxygen species were involved in the cytotoxicity induced by alginate upon UVA irradiation. It appeared that superoxide anion is not implicated     

Extracellular calcium does not contribute to cryopreservation-induced cytotoxicity

Summary The possible role of extracellular calcium ([Ca⁺²]e) in cryopreservation-induced cytotoxicity was tested using Madin-Darby canine kidney (MDCK) cells and a fluorescent multiple endpoint assay. MDCK cells maintained in 2 mM [Ca⁺²]e and treated with the calcium ionophore, ionomycin, increased their intracellular calcium ([Ca⁺²]i) as revealed by the calcium indicator dye, Fluo3 and the bottom-reading spectrofluorometer, CytoFluor 2300. The addition of 10 mM [ethylene bis (oxyethylenenitrilo)]-tetraacetic acid (EGTA) to the extracellular medium before treatment with ionomycin blocked this ionomycin-dependent increase in [Ca⁺²]i. A number of site and activity-specific fluorescent probes were surveyed to determine which indicator dye might best reveal the ionomycin-induced cytotoxic events during this increase in [Ca⁺²]i. Although most dyes changed their emission profiles in response to calcium, neutral red was found to best reflect the loss of [Ca⁺²]i homeostasis. The NR50 for a 15-min exposure to ionomycin in the presence of 2 mM [Ca⁺²]e was approximately 2μM ionomycin, but ionomycin had little apparent effect on neutral red retention when 10 mM EGTA was added to the extracellular medium. Thus it was clear that an increase in [Ca⁺²]i could be cytotoxic to MDCK cells and that neutral red could monitor this cytotoxic episode. To test if [Ca⁺²]e was similarly cytotoxic during cryopreservation, MDCK cells were subjected to cryopreservation in the presence of dimethylsulfoxide (DMSO). In contrast to previous studies, plasma membrane integrity, not lysosomal function, seemed to best correlate with cell survival subsequent to cryopreservation. In addition, decreasing [Ca⁺²]e had no discernable effect on the retention of plasma membrane indicator dyes, neutral red, or cell survival. It is concluded that a) plasma membrane indicator dyes, not neutral red, might be better indicators of cytotoxicity occurring during cryopreservation; b) DMSO might be toxic to lysosomes during cryopreservation of cultured cells; and c) although [Ca⁺²]e can contribute to cytotoxicity, the presence of [Ca⁺²]e might not influence cryopreservation-induced cytotoxicity.     

Cytotoxicity as measured by trypan blue as a potentially confounding variable in the in vitro alkaline elution/rat hepatocyte assay

Rat hepatocytes treated in vitro with A2RA, an angiotensin II receptor antagonist, displayed increased level of DNA-strand breaks as determined by alkaline elution, without an appreciable increase in cytotoxicity as determined by a trypan blue dye exclusion assay at harvest. The alkaline elution profile appeared to have two components: a rapidly eluting component detected in the first fraction collected (often associated with DNA from dead or dying cells), followed by a more slowly eluting component detected in the subsequent fractions. Further analysis of hepatocytes treated with A2RA by pulsed-field gel electrophoresis and neutral elution revealed significant levels of DNA double-strand breaks. Electron microscopy (EM) showed pronounced damage to mitochondria; although cell blebbing was seen using both EM and light microscopy, the plasma and nuclear membranes appeared intact when examined by EM. Cellular ATP levels decreased precipitously with increasing doses of A2RA, falling to less than 10% of control values at a dose of 0.213 mM A2RA, a concentration showing 100% relative viability by trypan blue at harvest. Thus, whereas in our experience trypan blue dye exclusion accurately reflects cytotoxicity induced by the majority of test agents, in this rather unusual case, trypan blue did not accurately reflect compound-induced cytotoxicity at harvest since there was no concurrent loss of membrane integrity. However, when hepatocytes treated with A2RA were incubated for either 3 h or 20 h in the absence of compound, a sharp, dose-dependent decline in viability was observed using trypan blue dye exclusion. Together with the initial, dose-dependent drop in the alkaline elution curve, these data suggest that the observed DNA double-strand breaks arose as a consequences of endonucleolytic DNA degradation associated with cytotoxicity, rather than by a direct compound-DNA interaction. Since DNA double-strand breaks behave under alkaline denaturing conditions as two single-strand breaks and can therefore produce increases in the alkaline-elution slope values, a necessary criteria for a valid positive result in this assay is that cytotoxicity by trypan blue dye exclusion will not be greater than 30%. Our data, however, indicate that interpretation of the elution assay as a test for genotoxicity can still be confounded by the failure of the trypan blue dye exclusion assay to reflect cytotoxicity in the unusual instance when there is no concurrent, immediate loss of membrane integrity.     

Assessement of the relation between the initial viability and the attachment of freshly isolated rat hepatocytes used for the in vivo/in vitro DNA repair assay (UDS)

Crucial steps of the in vivo/in vitro DNA repair assay (UDS) are the hepatocyte isolation procedure and the establishment of the hepatocyte cultures. Since the attachment of the isolated hepatocytes on the surface of the culture vessel is an essential prerequisite for the in vitro part of this assay to yield scorable autoradiograms, we assessed the relation between the initial viabilities of hepatocyte preparations and the resulting attachment efficiencies from 286 rats. The initial viability was determined by means of the trypan blue dye exclusion assay. The actual cell number was corrected for the viability and a constant number of 2.5 × 10⁵ viable cells were seeded into each well of gelatinized six-well dishes. The amount of adherent cells was determined after a 1.5-h attachment period using a recently described modification (Fautz et al., 1991) of the neutral red dye absorption assay. The attachment is described by the optical density at 540 nm obtained after the elution of neutral red from the adherent cells (OD₅₄₀ value).To facilitate a comparison of the data we divided the 286 animals into eight arbitrary viability groups. The mean values of the viability groups were 53.1, 62.2, 66.3, 68.4, 70.9, 73.6, 76.9, and 84.0% living cells. Although there was a great interindividual variation, the resulting mean OD₅₄₀ values were nearly uniform, about 0.5, in all eight groups, regardless of the initial viability of the hepatocytes.UDS data obtained from 46 animals treated with the positive control chemical 2-acetylaminofluorene demonstrated that there was no correlation between the in vitro DNA repair capacity and the initial viability or the attachment efficiency of the hepatocytes.Our results suggest that (i) great interindividual differences exist between the attachment of particular cell preparations with no regard to the initial viability, (ii) the correction of the cell number for viability leads to relatively uniform OD₅₄₀ mean values and (iii) for an in vivo/in vitro UDS assay even cell suspensions with relatively low viabilities can be used since they will yield adherent cultures which are capable of DNA repair synthesis. The latter item often allows a reduction in the number of animals required for this in vivo assay because it is not necessary to perform repeated experiments because of low viability preparations.     

Application of the chemiluminescent assay to cytotoxicity test: detection of menadione-catalyzed H2O2 production by viable cells.

Menadione-catalyzed H2O2 production by viable cells is proportional to viable cell number. The correlations between the viable cell number and the concentration of H2O2 produced are determined with the rapid chemiluminescent assay (S. Yamashoji, T. Ikeda, and K. Yamashoji, 1989, Anal. Biochem. 181, 149-152). This chemiluminescent assay of viable cells requires only 10 min and is much faster than NR (neutral red) inclusion and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assays, which require 3-5 h. When viable cells are incubated with antitumor drugs, detergents, mycotoxins, and glycoalkaloids for 24-48 h, a decrease in menadione-catalyzed H2O2 production in a dose- or incubation time-dependent manner is observed. In general, the 50% inhibition concentration determined by the chemiluminescent assay is lower than that determined by NR inclusion and MTT reduction assays, and the order of relative cytotoxic effects of agents is the same among these assays. Furthermore, clear cytotoxic effects are observed by the chemiluminescent assay after 1 h exposure of trypsinized cells to toxic compounds. Therefore, the chemiluminescent assay is expected to be more useful for the rapid detection of cytotoxic compounds than NR inclusion and MTT reduction assays.     

Potentiation of cytotoxic effect of lymphotoxin by anti-cancer drugs and elevated temperatures

The cytotoxic lymphokine, lymphotoxin (LT), has been shown to possess antitumor effect in vitro and in vivo. We examined the effect of the combination of partially purified LT with anti-cancer drugs and elevated temperatures on mouse transformed fibroblast cell line, L-929, and two human carcinoma of the cervix cell lines, HeLa and ME180. The cells were treated for 7 hr with Adriamycin, cisplatin, or bleomycin. These cells were then incubated for 24 hr in the presence of LT. At the end of the incubation period, cytotoxicity was measured by the neutral red dye uptake assay. There was 10- to 47-fold potentiation of cytotoxicity of LT on L-929 cells. The potentiation of cytotoxicity on human carcinoma of cervix cell lines ranged from 3- to 23-fold. L-929 cells and ME180 cells were incubated for 7 hr at 40 or 42 degrees C followed by 24 hr of incubation in the presence of LT. The elevated temperature treatment also enhanced (5- to 9-fold) the cytotoxic effect of LT. DNA, RNA, and protein syntheses of the ME180 cells was measured following incubation at 42 degrees C. It was observed that all three parameters were suppressed by incubation at this temperature. It was, therefore, possible that the repair of LT damaged cells was hampered by the elevated temperature treatment. It is suggested that LT may have a potential as an anti-tumor agent in combination with selected therapeutic drugs and hyperthermia.     

A model culture system with human gingival fibroblasts for evaluating the cytotoxicity of dental materials

Summary A model experimental culture system and protocol are described to screen polymerized dental materials for diffusible toxic products. The system employs cultures of human gingival fibroblasts grown in plates containing immobilized samples of polymerized resins. Comparative cytotoxicity is evaluated by counting viable cells with the aid of phase optics at several time periods up to 48 h. To achieve adequate statistical sampling, multiple counts are made in four different zones at 90° angles from each sample and at three distances from the centers of samples. The most significant data were generated during a 24 to 48 h test period in culture. This cytotoxicity test measured cell death as a function of time of exposure and distance from the sample (24 h, 0 to 3 mm; 48 h, 3 to 6 mm) and permitted a calculation of the relative cytotoxicity for each material, which is termed the viability index (VI). This can be expressed as a percentage related to the control, which is called the time-distance cytotoxicity index (TDCI). This method is simple to carry out because it uses basic laboratory equipment, is rapid, and has a sound scientific basis. It focuses on times and distances when or where, or both, the greatest cellular changes are taking place. Some data illustrated are based on the screening of eight different restorative resins. The literature of cell culture testing of dental materials is reviewed. It is concluded that biotoxicity studies ideally should employ diploid human target cells from the oral cavity because the cells retain specialized features. Secondary cultures or strains of human diploid gingival fibroblasts, which are relatively easy to obtain and maintain, are recommended as cells of choice for screening dental restorative materials in vitro. This project was supported in part by an intramural grant from the Louisiana State University School of Dentistry, derived from BRSG Grant S07-RR-05704-10 awarded by the Biomedical Research Grant Program, Division of Research Resources, National Institutes of Health, Bethesda, MD.     

Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay.The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC₅₀ values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity.Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC₅₀ values.     

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.     

Modified Field stain - rapid viability test for Trichomonas vaginalis

Aims: We previously reported that Modified Field Stain (MF) can be used as a rapid stain for diagnosis. In the present study we extend the observation to include the stain as an alternative method to assess viability of the cells. Methods and Results: Six isolates of Trichomonas vaginalis were used to assess the utility of the Modified Field stain as a rapid viability test for T. vaginalis and to compare with 0·4% Trypan Blue dye exclusion test in three conditions; normal in vitro culture growth using Hollander medium, lysed in distilled water and treated with metronidazole. MF stain showed similar growth profile pattern as Trypan Blue dye exclusion for identifying viable cells of T. vaginalis. Although, Trypan Blue dye exclusion test is ready made, rapid and widely used in laboratory as reliable viability assay, however, the limitation using Trypan Blue is the dye was unable to show internal morphological changes during the parasite’s transition from being viable to non‐viable. On day 3 where cultures peaked the correlation factor of both assays done to assess the viability of parasites harvested from the controls, metronidazole and distilled water treated parasites were more than 0·9 respectively. Conclusions: This confirms that MF staining does not only record permanently the morphological changes and retain internal structural details but also provides a reliable and rapid viability assay for the parasites. Significance and Impact of the Study: Therefore, in our study, Modified Field’s stain may offer the researchers and laboratory technologists the opportunity to get the result on the same day and the most important thing is the ability to differentiate between viable and non‐viable of T. vaginalis under three different conditions (normal culture, drug and distilled water condition). Modified Field’s staining method enhanced the morphological identification of T. vaginalis compared to Trypan Blue dye exclusion.     

A simple method for the quantitation of the stem cells derived from human exfoliated deciduous teeth using a luminescent cell viability assay

Stem cells from human exfoliated deciduous teeth are a population of highly proliferative postnatal stem cells and have been characterized as multipotent stem cells. In this study we developed a fast and sensitive method for stem cells derived from human exfoliated deciduous teeth count, using a luminescent viability assay. We isolated stem cells from normal exfoliated deciduous teeth using collagenase/dispase digestions solutions. Separated stem cells were placed in opaque-walled multiwall plates in culture alpha Modified Eagle’s Medium. For dental pulp stem cells quantitation we used a simple method for determining the number of viable cells based on ATP concentration. Cells attached to the bottom of the multiwall plates were counted with the luminescent assay and were cultured for mesenchymal markers expression. Moreover cells attached to the bottom of the multiwall plates were directed toward the osteogenic, adipogenic, lineages at the respective passages. Flow cytometry was used for immunophenotyping of cultured dental stem cells from exfoliated deciduous teeth. Cells that were counted with the luminescent assay, after culture formed fibroblastic morphology and were expressed the mesenchymal stem cell markers CD29, CD105, CD146, CD44. There was a correlation between the number of cells plated for culture and the number of mesenchymal stem cells after culture. Osteogenic and adipogenic differentiation of the cells counted with the luminescent assay was performed. The luminescent signal of viable mesenchymal dental stem cells isolated from dental pulp of exfoliated teeth represents an ideal method for mesenchymal stem cells count before culturing.     

Cytotoxicity of DMSO for MRC5, Chang liver and CV1 cells evaluated in vitro by LK, MTT and NR assays

Evaluation of chemicals cytotoxicity plays fundamental role in many in vitro investigations. The way of assessment of cytotoxicity depend on aim of study, characteristic of used cells and mode of action of investigated chemicals. The principal aspect of these investigations is validation of used method. In this paper validation of three different cytotoxicity assays is presented: total cell number measurement (LK), microplate assay measured mitochondrial dehydrogenase activity (MTT) and colorimetric assay measured ability of live cell to uptake neutral red (NR). The investigation was performed on different cells (MRC5, CV1 i Chang Liver) with DMSO as reference agent.