[Significance of tetrazolium salt based colorimetric assay (MTT assay) for isolated rabbit parietal cell].
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Animals↗
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Results for “MTT assay”
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[Study on chemosensitivity test (in vitro) of cultured human urogenital carcinoma cell lines by MTT assay].
MTT assay was performed simultaneously with a clonogenic assay to assess its validity on chemosensitivity test by using the cultured human urogenital carcinoma cell lines PC3, 19PC93, DU145 and T24. The drugs used were carboquone (CQ), adriamycin (ADM), cisplatin (CDDP) and pepleomycin (PLM). Dose response curves were drawn and 50% inhibition doses (ID50) were calculated and examined by t-test. The correlations between the results obtained from both assays were observed. For comparing antitumor intensity of drugs with each other, predicted antitumor activity (PAA) was calculated from the peak plasma concentration of the clinically used dose. High cytotoxicity of drug was considered if PAA greater than or equal to 1 was observed. The optical density (OD) was almost directly proportional to the number of cells. Good correlations between OD and colony number, or between ID50 from both assays were noted although the clonogenic assay is more sensitive than the MTT assay. Relative resistances between cell lines to a drug observed by the clonogenic assay were also maintained by the MTT assay. The chemosensitive intensity was CQ greater than ADM greater than CDDP greater than PLM, and the sequence was similar in both assays. PC3, DU145 and T24 were sensitive to CQ but 19PC93 was to CQ and ADM. Therefore, MTT assay was concluded as a useful method for chemosensitivity test, although the problem of normal cell contamination was left to be solved for clinical use.
Antineoplastic Agents↗
[Compared study on Fura-2/AM assay and MTT assay for screening multidrug resistant modulators].
To explore the difference of screening results of reversing multidrug resistance (MDR) by modulators between Fura-2/AM assay and MTT assay, 25 compounds which have active structure were studied for MDR reversal activity with both methods. The fold of MDR reversal was shown to have remarkable relation with the amount of Fura-2 accumulation (Y = -3.66 + 17.5X, gamma = 0.86, P < 0.01). On the other hand, Fura-2/AM assay has several advantages as compared with MTT assay. Fura-2/AM assay needs shorter time (4 h) than MTT assay (96 h), and the MTT assay needs more steps than the Fura-2/AM assay. Furthermore, Fura-2/AM assay was more reliable than MTT assay for screening MDR modulators because MTT assay was dependent on the viable cells, while Fura-2/AM assay was dependent on the function of P-gp. The results suggest that Fura-2/AM assay may replace MTT assay in the screening of MDR modulators on a large scale.
Bepridil↗
Comparison of chemosensitivity tests: clonogenic assay versus MTT assay.
When the development of chemotherapeutic agents reaches the clinical trial stage, it is necessary to perform drug sensitivity tests quickly in order to select the most promising agents for the treatment of cancer. In order to assess the possibility of using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as a substitute for the human tumor clonogenic assay (HTCA), we evaluated the correlation between the results obtained by these 2 assays in 5 human lung cancer cell lines. The correlation coefficient between the results of the HTCA and the MTT assay was 0.673, indicating a relatively good correlation. The correlation was most prominent in platinum analogues (r = 0.939) and good in anthracyclines/anthracenedione (r = 0.611). However, no significant correlation was observed in vinca alkaloids, etoposide, irinotecan, SN-38 (an active metabolite of irinotecan), and rhizoxin. The results of the MTT assay showed a high degree of correlation with those of the HTCA in predicting the sensitivity of cancer cell lines to platinum analogues, and anthracyclines/anthracenedione. These results suggest that the MTT assay may be more convenient and quickly performed than the HTCA and can replace HTCA in evaluating the effects of anticancer agents, especially the platinum analogues and anthracyclines/anthracenedione.
Antineoplastic Agents↗
The influence of stromal cells on the MTT assay (II)--Study on the nude mouse system.
A fresh surgical specimen from colon carcinoma was tested by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide assay (MTT assay) and transplanted into nude mice. After 5 transfers in male BALB/c nude mice, the xenograft was then tested again by the MTT assay. It was found that the in vivo chemosensitivity pattern of the xenograft was essentially identical to that of the in vitro fresh surgical specimen, whereas the sensitivity of the xenograft was increased. To exclude the stromal cells from the nude mouse, anti-BALB/c serum was added to the primarily cultured colon carcinoma xenograft, and its chemosensitivity to mitomycin C (MMC) assessed. Although the sensitivity of the serum-treated group to MMC was slightly higher than that of the untreated group, the dose-response curves of the tumor cells to MMC were similar to each other. Thus, the chemosensitivity pattern of tumor cells seemed to be stable with or without normal cells, although the sensitivity itself was reduced by the presence of normal cells.
Animals↗
Evaluation of MTT assay in agarose for chemosensitivity testing of human cancers: comparison with MTT assay.
The MTT assay in agarose, a simple colorimetric test performed in double-layer agarose, has been evaluated for chemosensitivity testing of fresh tumor samples from human cancers in comparison with the MTT assay. The absorbance of cells from fibroblast cell lines or normal tissues was markedly reduced in agarose. The chemosensitivity of cells from a carcinoma cell line or fresh tumor tissues was not apparently affected by the presence of almost 50% of fibroblast or nonmalignant cells in the MTT assay in agarose, whereas it did so in the MTT assay. The frequency of the differences between chemosensitivity of fresh tumor samples in both assays was increased, when the tumor tissue cells contained a higher proportion of nonmalignant cells, i.e. vimentin-positive cells. In 173 patients with various carcinomas, in vitro sensitivity to 7 drugs in the MTT assay in agarose was significantly greater than that in the MTT assay. Further, the MTT assay in agarose had a higher accuracy for prediction of either sensitivity or resistance than the MTT assay in a total of 38 in vitro-in vivo correlations. These results indicated that the MTT assay in agarose was a more suitable technique for the application to chemosensitivity of fresh tumor samples from patients with various carcinomas as compared to the MTT assay.
Adult↗
In vitro evaluation of cell/biomaterial interaction by MTT assay.
The tetrazolium-based colorimetric assay (MTT test) measures only in vitro living cells and the results are directly related to the number of viable cultured cells. It has been adopted in immunological investigations, cancer research and, recently, biocompatibility evaluation. We used the MTT method with minor modifications to fit it to an in vitro study of biomaterial-cell interactions. The MTT assay was confirmed to be feasible, rapid and reproducible. Moreover, it showed a good correlation with other in vitro proliferation assays, such as the 3H-thymidine uptake assay. By using the MTT method and the ASTM procedure for extracting biomaterials, we quantified the in vitro cell compatibility of different metals and polymers.
Biocompatible Materials↗
Chemosensitivity testing of primary human renal cell carcinoma by a tetrazolium based microculture assay (MTT).
MTT staining procedures have been used in chemosensitivity testing of established cell lines of human and other sources as well as of human leukaemias, but only limited information on its application in primary solid human tumors is presently available. We have evaluated MTT staining in primary human Renal Cell Carcinomas (RCCs), studied various factors interfering with the optimal use, and finally applied it in subsequent chemosensitivity testing. The method depends on the conversion of a water-soluble tetrazolium salt (MTT) to a purple colored formazan precipitate, a reaction effected by enzymes active only in living cells. Single cell suspensions of RCCs were obtained either by enzymatic dispersion or by mechanical dissagregation, filtered through gauze, and purified by Ficoll density centrifugation. Tests were carried out in 96-well microculture plates. 10(4) viable tumor cells per well at 4 h incubation time with 20 micrograms MTT/100 microliters total medium volume yielded best results. Formazan crystals were dissolved with DMSO, and the plates were immediately measured on a microculture plate reader at 540 nm. Under these criteria, linearity of the system could be demonstrated. For chemosensitivity testing, cells were continuously exposed to a number of drugs prior to the MTT staining procedure. Reproducibility of results was assessed and confirmed by culturing RCCs in flasks additionally, resubmitting them after 1, 2, and 4 weeks to the MTT assay. We conclude that the semiautomated MTT assay offers a valid, rapid, reliable and simple method to determine the degree of chemoresistance in primary human RCCs.
Antineoplastic Agents↗
The influence of stromal cells on the MTT assay (I)--In vitro chemosensitivity of the tumor and stromal cells to mitomycin C.
To clarify the influence of stromal cells on the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide assay (MTT assay), a gastric carcinoma cell line (KATO-III) and a human fibroblast cell line (IMR-90) were subjected to a colorimetric assay, in which the chemosensitivity KATO-III was found to be highly sensitive to mitomycin C at 10 micrograms/ml, whereas IMR-90 was insensitive to mitomycin C at the same concentration. When the mixtures of these two cell lines were tested by the assay, a mixture of more than 25 per cent stromal cells reduced the sensitivity of KATO-III to mitomycin C. This suggested that the stromal cells in fresh surgical specimens might reduce the apparent sensitivity of the tumor cells.
Drug Screening Assays, Antitumor↗
Cell sensitivity assays: the MTT assay.
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Animals↗
Enhanced chemosensitivity in acute myeloid leukemia by hematopoietic growth factors: a comparison of the MTT assay with a clonogenic assay.
The MTT assay, a colorimetric assay, is found to be suitable for chemosensitivity testing. Recently, it has been suggested that hematopoietic growth factors (HGF) may enhance the effects of cytostatic drugs in acute myeloid leukemia (AML). We therefore studied the effects of granulocyte colony-stimulating factor (G-CSF), interleukin 3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) combined with cytosine arabinoside (Ara-C), daunorubicin (DNR), mitoxantrone (MXT), or etoposide (VP-16) by using the MTT assay. The results were compared with in vitro clonogenic assays. Briefly, AML cells of nine patients were incubated in the presence or absence of G-CSF, IL-3, or GM-CSF under serum-free conditions for 24 hours. Next, for the MTT assay, Ara-C (final dilution range: 0.0024-240 micrograms/ml), DNR (final dilution range: 0.05-3.2 micrograms/ml), MXT (final dilution range: 0.05-3.2 micrograms/ml), or VP-16 (final dilution range: 0.1-100 micrograms/ml) were added and incubated for 48 hours. Cell survival was determined and IC75 values (75% reduction as compared to control cultures) were calculated. For clonogenic assays, the three lowest drug concentrations were used. After 48 hours, the clonogenic response was determined in serum-free, semi-solid cultures with G-CSF, IL-3, or GM-CSF. The results obtained by the MTT assay showed no significant enhancement of cytotoxicity by HGF on cytostatic drug preincubated cells compared to cytostatic drugs alone. The results obtained by the clonogenic assays showed increased cytotoxicity of Ara-C combined with G-CSF, IL-3, or GM-CSF. The median IC75 values of Ara-C decreased from 0.056 to 0.0168 microgram/ml with G-CSF (p = 0.01), from 0.108 to 0.0168 microgram/ml with IL-3 (p = 0.004) and from 0.12 to 0.0204 microgram/ml for GM-CSF (p = 0.02). Only moderate enhanced cytotoxicity was observed when VP-16 was combined with IL-3 (p = 0.036) or GM-CSF (p = 0.036), but not with G-CSF. No enhanced cytotoxicity of DNR and MXT to clonogenic AML cells was found when these agents were combined with HGF stimulation. The results indicate that the MTT assay underestimates HGF enhanced cytotoxicity of Ara-C or VP-16 to clonogenic cells. Therefore, the assay is not useful for accurately detecting differences of clonogenic response due to the proliferative status of cells. In this paper, the potential explanations for the failure of the MTT assay are discussed.
Adult↗
Predictability of in vivo chemosensitivity by in vitro MTT assay with reference to the clonogenic assay.
The MTT assay reported by Mosmann is a rapid and convenient colorimetric assay for cellular growth and survival in vitro. In this paper, the MTT assay was modified as a chemosensitivity test, and its potential was investigated. Using 10 human tumor xenografts in athymic nude mice, the predictability of in vitro antitumor effects of drugs using the MTT assay was compared with that using the clonogenic assay. The MTT assay showed excellent reproducibility, and the predictable rate in this assay was 86.7%, with 100% true-positive and 77.8% true-negative rates, almost equivalent to the 90.0% predictable rate of the clonogenic assay. This method also has several advantages with respect to rapidity, quantitation, management of many samples, and cell number required for the assay. Application of this assay to chemosensitivity testing seems to be valuable and useful.
Animals↗
An improved MTT assay.
The MTT assay for cell viability and cell proliferation has been modified to improve its reproducibility and accuracy. The modified test is performed on MultiScreen filtration plates, which permits the removal of the culture medium prior to formazan solubilisation, without loss of cells or formazan crystals. A 1:1 mix of DMSO and ethanol is used as the solvent, since this has the same optical refraction index as the filters, making it possible to measure the optical densities directly on the MultiScreen plate. Data obtained in the assay method using the MultiScreen plate were compared with data from studies employing the normal flat-bottomed plate, by using cells which grow in suspension. Two cell lines were used in the study. CTLL-2, which are IL-2 dependent cells of murine T cell origin, and Jurkat E.6.1 which are IL-2 producing cells of human lymphoma origin. CTLL-2 cells and the modified MTT assay were also used for evaluating the effects of different IL-2 concentrations on cell proliferation.
Animals↗
A modified computer-assisted colorimetric microtitre assay (MTT) to assess in vitro radiosensitivity of V79, CaSki, HeLa and WiDr cells.
The non-clonogenic MTT assay, based on the reduction of a tetrazolium salt to a purple formazan precipitate by living cells, was modified and a new procedure of analysis is proposed. The colorimetric assay could be performed semi-automatically using microtitre plate reader connected to a personal computer. Data are processed and plotted with a customized program. To ensure that both control and irradiated microtitre plates contain exponentially growing cells at the time of analysis, the calculation of relative survival is based on a series of well-defined cell numbers initially seeded. To make the two endpoints of non-clonogenic and clonogenic assays comparable, i.e. counting of living cells versus counting of colonies, radiation-induced progression delay was incorporated into the calculation. Radiation-induced cell killing and progression delay could be determined in a single analysis, but in an independent way. X-ray survival curves were generated for V79, CaSki, WiDr and HeLa cells using the non-clonogenic and a standard clonogenic assay. Using the linear-quadratic formula, the resulting parameters alpha, beta and the mean inactivation dose were not significantly different. The described assay is a feasible and reproducible technique for determination of cellular survival, which may be able to incorporate progression delay. The equivalence to a clonogenic survival assay could be proven.
Animals↗
[Chemosensitivity testing on human bladder cancer cell lines, using MTT-assay].
Sensitivity of human transitional cancer cells to anticancer agents was evaluated utilizing cultured cell lines. T-24, MGH-U1 and KU-1. Simultaneously, chemosensitivity tests combined with 42 degrees C hyperthermia were performed. Cells inoculated in 96-well multiplates for 48 hours, were exposed to graded concentrations of doxorubicin (DOX), mitomycin C (MMC), bleomycin (BLM), peplomycin (PEP), cis-diamminedichloroplatinum (II) (CDDP) for 2 to 48 hours. After additional culture for 48 hours, viable cell numbers were estimated by the dye exclusion assay (DEA) and tetrazolium-based colorimetric assay (MTT-assay). In 2-hour exposure, most of anti-cancer agents did not significantly suppress the growth of the cell lines. Only DOX suppressed the cell growth. In 6-hour and 48-hour exposure, DOX, MMC and CDDP showed significant growth inhibitory effect on the transitional cancer cell lines. The effect of BLM and PEP was insufficient. The hyperthermia of 42 degrees C enhanced the growth inhibitory effect of MMC and CDDP, but did not influence the effect of DOX. In comparison of DEA and MTT-assay, viable cell numbers measured by DEA well correlated with the optical density in MTT-assay. Since MTT-assay is a semiautomated, rapid and inexpensive assay with good reproducibility, it can be a useful substitute for DEA in chemosensitivity testing of cancer cells.
Antineoplastic Agents↗
Differential methotrexate resistance in childhood T- versus common/preB-acute lymphoblastic leukemia can be measured by an in situ thymidylate synthase inhibition assay, but not by the MTT assay.
Methotrexate (MTX) is not cytotoxic to patient-derived acute lymphoblastic leukemia (ALL) cells in total-cell-kill assays, such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, putatively due to the rescue effects of hypoxanthine and thymidine released from dying cells. This was mimicked by a diminished methotrexate (MTX) cytotoxicity for the cell lines HL60 and U937 in the presence of hypoxanthine, thymidine, or lysed ALL cells. However, enzymatic depletion or inhibition of nucleoside membrane transport did not result in MTX dose-dependent cytotoxicity in patient samples. Alternatively, a thymidylate synthase inhibition assay (TSIA), based on inhibition of the TS-catalyzed conversion of 3H-dUMP to dTMP and 3H2O, correlated with the MTT assay for antifolate sensitivity in four human leukemia cell lines with different modes of MTX resistance. For 86 ALL patient samples, TSI50 values after 21 hours exposure to MTX were not different between T- and c/preB-ALL (P =.46). After 3 hours incubation with MTX followed by an 18-hour drug-free period, T-ALL samples were 3.4-fold more resistant to MTX compared with c/preB-ALL samples (P =.001) reflecting the clinical differences in MTX sensitivity. TSI50 values correlated with MTX accumulation (r = -.58, P <.001). In conclusion, the TSIA, but not the MTT assay, can measure dose-response curves for MTX in patient-derived ALL cells and showed relative MTX resistance in T-ALL compared with c/preB-ALL.
Adolescent↗
MTT-assay and neutral red release (NRR)-assay: relative role in the prediction of the irritancy potential of surfactants.
A comparative study on the in vitro and in vivo irritancy of anionic, amphoteric and non-ionic surfactants was performed. In vitro ED50 values of the surfactants were determined by two cytotoxicity assays, the dimethylthiazoldiphenyltetrazoliumbromide (MTT) assay and the neutral red release (NRR) assay on serum-free cultured human foreskin keratinocytes. In vivo human irritancy data were obtained by a 24 hour occlusive patch test in volunteers and the irritant skin response quantified by visual scoring, evaporimetry and colorimetry. A close relationship between the evaluation methods of the patch test was observed (r = 0.92 to r = 0.96), confirming that the 'bioengineering' methods, such as evaporimetry and colorimetry are suitable for measuring skin irritation. For six surfactants evaluated we found a good correlation (r = 0.91) between the ED50 values of the MTT assay and the in vivo irritancy data. The NRR assay yielded less satisfactory correlation coefficients with regard to MTT assay (r = 0.42) and in vivo irritancy data (r = 0.46). This can be mainly attributed to a misinterpretation of the amphoteric and non-ionic surfactants by the NRR assay. While the NRR assay may better evaluate the anionic surfactants, the MTT assay seems to be more suitable when testing a broader range of chemically diverse surfactants. Limitations of cell culture systems are noted, although the potential usefulness of cultured human skin cells for skin irritancy testing has been clearly demonstrated.
Adult↗
[Sensitivity test for anti-tumor agents-3. MTT assay and its clinical effect].
MTT assay, a sensitivity test of anti-tumor agents was performed, and its clinical usefulness, was discussed. In 15 surgical specimens, the cell suspensions were prepared aseptically, and divided into three processing; namely, Papanicolaou (Pap) smears to confirm the malignant cells, MTT assay, and cell cultures in chamber slides. MTT assay was evaluated only when tumor cells in the chamber slide were observed 50% or more by Pap staining. Drugs judged to be effective were applied for patients, resulting in 64.2% of predictive accuracy in the sensitivity. Conclusions; 1) MTT assay was developed for sensitivity test of anti-tumor agents, 2) Strict assessment was carried out by the confirmation of cancer cells using chamber slides, 3) On clinical usefulness, predictability for the sensitivity was 64.2%, that for resistance was 100%, and over all predictability was 66.7% 4) MTT assay was useful for the determination of effective and for elimination of ineffective drugs.
Antineoplastic Agents↗