WEHI164－C1细胞株检测TNF的MTS／PMS比色法的建立

本文用有限稀释法对ＷＥＨＩ１６４细胞进行了亚克隆，从１２株中筛选出一株对ＴＮＦ高度敏感的亚克隆细胞株ＷＥＨＩ－Ｃｌ。ＭＴｓ的还原产物具有良好的水溶性，ＭＴＳ／ＰＭＳ比色法与ＭＴＴ法相比较具有简便、快速的优点。我们首先将ＭＴＳ引入细胞毒的检测，用ＷＥＨＩ１６４－Ｃｌ亚克隆株建立了检测ＴＮＦ的ＭＴＳ／ＰＭＳ比色法。此方法不仅简便、快速．而且敏感度高（比常规Ｌ９２９细胞检测法敏感１０～１５倍）、特异性强（ＩＬ－１、ＩＬ－２、ＩＬ－６、ＰＨＡ、ＣｏｎＡ、ＬＰＳ对检测结果均无明显影响）。     

WEHI164—C1细胞株检测TNF的MTS／PMS比色法的建立

本文用有限稀释法对ＷＥＨＩ１６４细胞进行进行了亚克隆，从１２株中筛选出一株对ＴＮＦ高度敏感的亚克隆细胞株ＷＥＨＩ－Ｃｌ０ＭＴＳ的还原产物具有良好的水溶性，ＭＴＳ／ＰＭＳ比色法与ＭＴＴ法相比较具有简便、快速的优点。我们首先将ＭＴＳ引入细胞毒的检测，用ＷＥＨＩ１６４－Ｃ１亚克隆株建立了检测ＴＮＦ的ＭＴＳ／ＰＭＳ比色法。此方法不仅简便、快速，而且敏感度高（比常规Ｌ９２９细胞检测法敏感１０－１５倍）、特异     

雷公藤T4组分对人脐带静脉内皮细胞增殖及产生IL—6…

本文报道雷公藤Ｔ４组分对血管内皮细胞增殖及产生白细胞介素－６（ＩＬ－６）的影响研究，将人脐带静脉血管的皮细胞与Ｔ４不同剂量（２．５，５，１０和２０ｎｇ／ｍｌ）一起培养２４小时，ＭＴＴ比色法测定发现，Ｔ４浓度为２０ｎｇ／ｍｌ时显著抑制内皮细胞的增殖（Ｐ＜０．０５）；经ＥＬＩＳＡ法显示，Ｔ４浓度５ｎｇ／ｍｌ时始显著抑制白皮细胞产生ＩＬ－６（Ｐ＜０．０５）。这些抑制作用与Ｔ４剂量呈依赖趋势。     

雷公藤T4组分对人脐带静脉内皮细胞增殖及产生IL－6的影响

本文报道雷公藤Ｔ４组分对血管内皮细胞增殖及产生白细胞介素－６（ＩＬ－６）的影响研究，将人脐带静脉血管内皮细胞与Ｔ４不同剂量（２．５，５，１０和２０ｎｇ／ｍｌ）一起培养２４小时，ＭＴＴ比色法测定发现，Ｔ４浓度为２０ｎｇ／ｍｌ时显著抑制内皮细胞的增殖（Ｐ＜０．０５），经ＥＬＩＳＡ法显示，Ｔ４浓度５ｎｇ／ｍｌ时始显著抑制内皮细胞产生ＩＬ－６（Ｐ＜０．０５）。这些抑制作用与Ｔ４剂量呈依赖趋势。     

MTT比色法检测IL—2和杀伤细胞活性的研究

本文采用ＭＴＴ比色法测定了ＬＡＫ和ＴＩＬ细胞培养上清ＩＬ－２活性及ＬＡＫ细胞介导对体外培养的白血病细胞株的杀伤活性。结果表明ＬＡＫ细胞和ＴＩＬ细胞培养上清有较高的ＩＬ－２活性，可用于支持培养ＩＬ－２依赖株的生长；ＬＡＫ细胞对体外培养的白血病细胞株有明显的杀伤作用。其杀伤效应随效靶比例增加而增高；同时亦证明：活的白血病细胞数与ＭＴＴ甲赞形成产物呈直线相关性，提示通过ＭＴＴ法测定ＯＤ值能较好地反映活细     

小鼠胸腺上皮细胞株MTECB自发产生低水平IL—6

本室所建立的小鼠胸腺上皮细胞株ＭＴＥＣＢ以５．０×１０＾５细胞／ｍｌ的浓度在含１０％小牛血清的ＤＭＥ培养液中，５％ＣＯ２３７℃条件下，培养６ｄ，离心收集上清液。以ＩＬ－６依赖细胞株采用ＭＴＴ法对ＭＴＥＣＢ的上清液进行ＩＬ－６活性检测。结果发现ＭＴＥＣＢ能自发产生低水平的ＩＬ－６，其活性单位为１９．２Ｕ／ｍｌ。ＭＴＥＣＢ的ＩＬ－６产生量低于献报道的其他胸腺上皮细胞株，说明我们建立的胸腺上皮细胞株有     

用维生素K带电试剂改良的MTT测定法

用维生素Ｋ带电试剂改良的ＭＴＴ测定法〔英〕／ＨａｌｙａｒＧ…／／ＪｏｕｒｎａｌｏｌＩｍｍｕｎｏｌｏｇｉｃａｌＭｅｔｈｏｄｓ．－１９９４，１６８．～２５３ＭＴＴ测定法是利用活细胞线粒体酶把黄色的ＭＴＴ还原成兰色的甲，广泛地用于检测细胞代谢的能力、肿瘤细...     

TLSFJM对PMA诱导Jurkat细胞IL—2Ra链基因表达的抑制作用

本文应用原位杂交和双ＭｃＡｂ夹心ＥＬＩＳＡ方法，探讨了来自ＪＭＴ细胞白血病细胞系产生的抑制因子（ＴＬＳＦＪＭ）对ＰＭＡ诱导的Ｊｕｒｋａｔ细胞ＩＬ－２Ｒａ链基因表达的影响。结果表明：ＴＬＳＦＪＭ可明显抑制ＰＭＡ诱导的ｊｕｒｋａｔ细胞ＩＬ－２Ｒａ链ｍＲＮＡ转录，并降低膜表面ＩＬ－２Ｒａ链ｍＲＮＡ转录，并降低膜表面ＩＬ－２Ｒａ链的表达和细胞培养上清中可溶性ＩＬ－２Ｒ（ｓＩＬ－２Ｒ）水平。     

白血病患者骨髓移植后血清中IL—6变化的初步观察

采用白细胞介素－６（ＩＬ－６）依赖细胞（Ｂ９细胞）增殖ＭＴＴ比色分析，研究了５例自体骨髓移植（ＡＢＭＴ）患者血清中ＩＬ－６的活性。处于完全缓解期的急性粒细胞性白血病（ＡＭＬ）患者血清中ＩＬ－６活性显著地高于正常成人组。ＢＭＴ０ｄ时，ＩＬ－６活性显著地高于ＢＭＴ前水平；ＢＭＴ后第９￣１０ｄ时，ＩＬ－６活性明显地高于ＢＭＴ前和ＢＭＴ０ｄ，高于ＢＭＴ后２１ｄ和３５ｄ。ＢＭＴ后２１ｄ时，ＩＬ－６活性略高Ｂ     

TLSF_（JM）对PMA诱导Jurkat细胞IL－2Rα链基因表达的抑制作用

本文应用原位杂交和双ＭｃＡｂ夹心ＥＬＩＳＡ方法，探讨了来自ＪＭＴ细胞白血病细胞系产生的抑制因子（ＴＬＳＦ_（ＪＭ））对ＰＭＡ诱导的Ｊｕｒｋａｔ细胞ＩＬ－２Ｒα链基因表达的影响。结果表明：ＴＬＳＦ_（ＪＭ）可明显抑制ＰＭＡ诱导的Ｊｕｒｋａｔ细胞ＩＬ－２Ｒα链ｍＲＮＡ转录，并降低膜表面ＩＬ－２Ｒα链的表达和细胞培养上清中可溶性ＩＬ－２Ｒ（ｓＩＬ－２Ｒ）水平。     

Evaluation of MTS, XTT, MTT and3HTdR incorporation for assessing hepatocyte density, viability and proliferation

The new 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) colorimetric assays for assessing hepatocyte density, viability and proliferation were evaluated and compared with 3-(4,5-dimethlthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and tritiated thymidine (³HTdR) incorporation. OD values of MTS or XTT, which are metabolically reduced in viable cells to a watersoluble formazan product and can be read directly, had a good correlation coefficient with hepatocyte densities in a range of 2.5–40×10⁴ cells/ml (MTSr=0.952; XTTr=0.902) and with hepatocyte viability (MTSr=0.974; XTTr=0.975). At 0, 20, 40 and 60 hr cultures, the correlation coefficients with³HTdR for assessing hepatocyte viability and proliferation (MTSr=0.942–0.981; XTTr=0.953–0.992) were excellent. In contrast with MTS and XTT, MTT OD values had a poor correlation coefficient with hepatocyte densities (r=0.672), viability (r=0.622) and³HTdR incorporation (r=0.701–0.818 at 0, 20 and 40 hour cultures). This study shows that the MTS or XTT colorimetric assays for assessing hepatocyte density, viability and proliferation are more accurate, reliable and simpler than the widely used MTT assay. They avoid use of radioactive material as required for³HTdR incorporation. Of the two, the MTS colorimetric assay is more sensitive than the XTT.     

An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT

A new tetrazolium salt XTT, sodium 3'-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6- nitro)benzene-sulfonic acid hydrate, was evaluated for use in a colorimetric assay for cell viability and proliferation by normal activated T cells and several cytokine dependent cell lines. Cleavage of XTT by dehydrogenase enzymes of metabolically active cells yields a highly colored formazan product which is water soluble. This feature obviates the need for formazan crystal solubilization prior to absorbance measurements, as required when using other tetrazolium salts such as MTT. Bioreduction of XTT by all the murine cells examined was not particularly efficient, but could be potentiated by addition of electron coupling agents such as phenazine methosulfate (PMS) or menadione (MEN). Optimal concentrations of PMS or MEN were determined for the metabolism of XTT by the T cell lines HT-2 and 11.6, NFS-60 a myeloid leukemia, MC/9 a mast cell line and mitogen activated splenic T cells. When used in combination with PMS, each of these cells generated higher formazan absorbance values with XTT than were observed with MTT. Thus the use of XTT in colorimetric proliferation assays offer significant advantages over MTT, resulting from reduced assay time and sample handling, while offering equivalent sensitivity.     

MTT比色法检测IL－2和杀伤细胞活性的研究

本文采用ＭＴＴ比色法测定了ＬＡＫ和ＴＩＬ细胞培养上清ＩＬ－２活性及ＬＡＫ细胞介导对体外培养的白血病细胞株的杀伤活性。结果表明ＬＡＫ细胞和ＴＩＬ细胞培养上清有较高的ＩＬ－２活性，可用于支持培养ＩＬ－２依赖株的生长；ＬＡＫ细胞对体外培养的白血病细胞株有明显的杀伤作用。其杀伤效应随效靶比例增加而增高；同时亦证明：活的白血病细胞数与ＭＴＴ甲赞形成产物呈直线相关性，提示通过ＭＴＴ法测定ＯＤ值能较好地反映活细胞数目及细胞毒效应。     

Synchronous gastric and sebaceous cancers,a rare manifestation of MLH1-related Muir-Torre syndrome

Muir-Torre syndrome (MTS), a rare variant of the hereditary non polyposis colorectal cancer syndrome, is an autosomal dominant genodermatosis characterised by coincidence of sebaceous gland neoplasms (sebaceous adenoma, epithelioma, or carcinoma) and at least one internal malignancy. The underlying cause of MTS is a germline mutation in DNA mismatch repair genes MSH2, MLH1 and MSH6. We report the case of a 52-year-old caucasian woman with the development of metachronous colon cancer at the age of 38 years, uterine cancer at the age of 43 years, and unique occurrence of synchronous gastric and sebaceous carcinomas related to germline point mutation c. 2194A>T in the last exon of MLH1 gene, resulting in truncated protein in C-terminal region p. Lys732X due to premature stop codon. This mutation, not previously reported in MTS, disrupts the function of MutL complexes presumably by preventing the interaction with PMS1/PMS2 and impairing the endonuclease active site. This case points out the importance of sebaceous neoplasia, especially sebaceous adenocarcinoma, as cutaneous markers of MTS for timely implementation of cancer screening programs.     

Regulatory effect of e2, IL-6 and IL-8 on the growth of epithelial ovarian cancer cells

To determine the regulatory effects of estrogen and cytokine IL-6 and IL-8 on the growth of epithelial ovarian cancer （OVCA）, we first examined the status of estrogen receptors （ERα and ERβ）, IL-6 receptor （IL-6Rα and gp130）, and IL-8 receptor （IL-8RA and IL-8RB） on five epithelial OVCA cell lines by semiquantitative RT-PCR and Western blot analysis. Results showed that the expressions of these receptors were variable on the five cells. Those OVCA ceUs expressing the receptors were selected to study related molecular mechanism. MTT assay was performed to observe the effects of 17β-estradiol （E2）, IL-6 and IL-8 on cell proliferation. We discovered that E2 markedly promoted the proliferation of CAOV-3 and OVCAR-3 cell in a time- and dose-dependent manner. Tamoxifen （Txf）, an ER inhibitor, completely blocked the proliferation of the E2-induced cells, and IL-6- or/and IL-8-neutralizing antibody only showed partially blocking activity. IL-6 and IL-8 were able to significantly stimulate CAOV-3 and OVCAR-3 cell proliferation in a time- and dose-dependent manner, which had a potential synergistic effect on CAOV-3 cells but not on OVCAR-3 cells. The cell proliferation induced by these two cytokines was abolished completely by their specific neutralizing antibodies, partially by Txf, but not by unrelated goat IgG. Taken together, our results suggested that estrogen, IL-6 and IL-8 could modulate OVCA growth by forming a reciprocal cascade with amplifying effect.     

Current methodology of MTT assay in bacteria – A review

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium assay is a popular tool in estimating the metabolic activity of living cells. The test is based on enzymatic reduction of the lightly colored tetrazolium salt to its formazan of intense purple-blue color, which can be quantified spectrophotometrically. Under properly optimized conditions the obtained absorbance value is directly proportional to the number of living cells. Originally, the MTT assay was devised for use in eukaryotic cells lines and later applied for bacteria and fungi. As the mechanism of MTT reduction was studied in detail mostly considering eukaryotic cells, the lack of information resulted in generating a vast variety of MTT based protocols for bacterial enzymatic activity evaluation. In the presented article the main aspects of the MTT assay applicability in bacterial research were summarized, with special emphasis on sources of inaccuracies and misinterpretation of the test results.     

Paradoxical proliferative potential of iron (II) sulphate on cancer cells after the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay

There are several scientific approaches for the determination of cellular growth influences of known or novel substances under in vitro conditions, among which colourimetric absorption measurement is considered to be one of the convenient methods. [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) assay is one of the commonly used colourimetric absorption assays based on the ability of dehydrogenase from viable cells to produce the brown soluble formazan detectable at 490 nm. Here we have tested the possible growth influence of iron (II) sulphate on two human cancer cell lines, the K562 chronic myelogenous leukaemia and T47D breast carcinoma cells, based on the MTS assay. We found that iron (II) sulphate possessed an inhibitory effect when added at 16- to 125-microM concentrations, but iron (II) sulphate became growth stimulatory when its concentration was further increased to 1000 microM. In addition, a dose-dependent increase in absorbance at the same wavelength was observed when we repeated the experiments without the addition of MTS and phenazine methosulfate. When we further repeated the cell growth determinations using adenosine triphosphate content assay for K562 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for T47D, iron (II) sulphate showed a consistent dose-dependent growth inhibitory effect. Morphological investigation after methylene blue staining clearly demonstrated that iron (II) sulphate, at a concentration of 1000 microM, is cytotoxic to T47D cells. Interestingly, a consistent increment for the absorbance at 490 nm was further observed with increased iron (II) sulphate concentration either in the presence or absence of MTS even in a cell-free environment. Thus we conclude that iron (II) sulphate is actually growth inhibitory and even cytotoxic at high concentrations towards the K562 and T47D cancer cells and the paradoxical proliferative activity of iron (II) sulphate on these two cancer cell lines using the MTS assay was solely due to the oxidation of initial pale green iron (II) to brownish iron (III) during incubation in the aqueous condition.     

Optimization and validation of a colorimetric assay for Tetrahymena sp. survival

A colorimetric assay based on the reduction of 3-(4,5-dimethyl-2-thiazol-2-yl)-2,5-diphenyl-2h-tetrazolium bromide (MTT), for the quantification of Tetrahymena sp. survival is described. An increase in the concentration of Tetrahymena sp. cells from 0 to 1 x 10(6) cells/ml produced a linear (R(2)=0.9965) increase in the optical density (OD, 570-630 nm), and dead cells (pre-exposed to 250 mg/l formalin for 4 h) did not produce a background reading. Cells exposed to sublethal concentrations to formalin (100 mg/l or less for 4 h) recovered their growth. Using the MTT assay, we determined that Tetrahymena sp. is sensitive to formalin, chloramine-T, hydrogen peroxide, copper sulfate and NaCl. The sensitivity increased with increasing chemical concentrations and exposure time. Tetrahymena sp. was resistant to bromex and malachite green. The use of this assay in drug screening for the development of treatments for tetrahymenosis and as a bioassay to evaluate the toxicity of environmental toxicants is discussed.     

Setting of a colorimetric method to determine the viability of Trypanosoma cruzi epimastigotes

The method most commonly used in screening of drugs for the treatment of Chagas' disease, microscopic counting of viable trypanosomes, is time-consuming, labor-intensive, and dependent on the observer. Although the tetrazolium dye [MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is comparatively quick and accurate, it requires careful attention in design as well as in interpretation of the results. Therefore, we examined under various conditions the sensitivity and specificity of the MTT assay versus microscopic counting for determination of the viability of Trypanosoma cruzi for drug-screening purposes. We tested different concentrations of MTT in phenazine methosulfate (PMS) against T. cruzi epimastigotes of the Y strain in different stages of logarithmic growth. In our model, in tests of benznidazole and nifurtimox the optimal concentration of MTT was 2.5 mg/ml of PMS and the optimal incubation period was 75 min. This method detected parasite concentrations of approx. 500,000 epimastigotes/ml (P < 0.01), and the linear correlation between absorbance values and numbers of epimastigotes per milliliter was very strong (approx. R=0.99). The present MTT assay results in faster determination of the activity of compounds, is more objective, and enables testing of several drugs simultaneously. Received: 8 February 2000 / Accepted: 5 June 2000