XTT比色法测定细胞生长曲线

目的:研究XTT比色法用于细胞生长曲线检测的可行性.方法:采用XTT比色法检测卵巢癌细胞株3AO的细胞生长曲线,并与MTT法相比较.结果:XTT比色法检测获得的3AO细胞生长曲线与应用MTT法获得的细胞生长曲线大致相同,且比MTT法更加快速简便.结论:XTT比色法不仅可替代MTT法用于细胞生长曲线的检测,而且优于MTT法.     

XTT法和MTT法测定B淋巴细胞株细胞活性的实验研究

目的：比较两种常用的细胞活性检测方法MTT和XTT法间的异同。方法：以B淋巴细胞株Daudi细胞株为观察对象,分别采用MTT法和XTT法测出各自的OD值、抑制率及IC50,比较IC50敏感性及变异系数的差异。结果：MTT法和XTT法所求OD值变异系数无统计学差异;所求IC50分别为14.45μg/ml和11.24μg/ml,XTT法更敏感（P=0.031）。结论：MTT法价格便宜;XTT法价格较贵,但灵敏度更高,结果更稳定。     

姜黄素抑制子宫颈癌HeLa细胞的作用

目的：探讨姜黄素抑制子宫颈癌HeLa细胞的作用及其机制。方法以细胞培养，镜下观察细胞形态学改变和计数法测定生长曲线；用3H-脱氧胸苷掺入法测定对DNA合成的影响，以MTT比色法检测给药后HeLa细胞的增殖抑制情况。结果姜黄素作用HeLa细胞后，癌细胞生长延缓并萎缩，胞质粗糙，有大量颗粒状物堆积，而且药物浓度越大，形态学改变越明显；生长曲线测定、3H-脱氧胸苷掺入法及MTT比色法实验结果显示，姜黄素对HeLa细胞的增殖和生长有显著的抑制作用并呈明显的时间一剂量依赖关系，当姜黄素浓度为20μg／ml以上时，其对HeLa细胞的掺入抑制率高于5一Fu20μg／ml对该细胞的掺入抑制率。结论姜黄素对HeLa细胞具有直接杀伤作用，其机制可能通过干扰细胞代谢，改变细胞外暌的性质抑制肿瘤细胞增值。     

姜黄素对子宫颈癌HeLa细胞的抑制作用

目的：探讨姜黄素对子宫颈癌HeLa细胞的抑制作用及其机制。方法：以细胞培养，镜下观察细胞形态学改变和计数法测定生长曲线；用^3H-脱氧胸苷掺入法测定对DNA合成的影响，以MTT比色法检测给药后HeLa细胞的增殖抑制情况。结果：姜黄素作用HeLa细胞后，癌细胞生长延缓并萎缩，胞质粗糙，有大量颗粒状物堆积，而且药物浓度越大，形态学改变越明显；生长曲线测定、^3H-脱氧胸苷掺入法及MTT比色法实验结果显示，姜黄素对HeLa细胞的增殖和生长有显著的抑制作用并呈明显的时间一剂量依赖关系，当姜黄素浓度为20μg／ml以上时，其对HeLa细胞的掺入抑制率高于5-Fu 20μg/ml对该细胞的掺入抑制率。结论：姜黄素对HeLa细胞具有直接杀伤作用，其机制可能通过干扰细胞代谢，改变细胞外膜的性质抑制肿瘤细胞增殖。     

过氧化氢对FRTL细胞线粒体膜电位和超氧化物生成的影响

目的:探讨外源性过氧化氢(H2O2)对Fisher大鼠甲状腺细胞系(FRTL)线粒体膜电位(△ψ)和超氧化物生成的影响.方法:用1 mmol/L H_2O_2处理FRTL细胞10 min、30 min、24 h后,利用MitoSOX,通过活细胞影像法、流式细胞术检测线粒体超氧化物生成;利用罗丹明123(rh123),通过荧光分光光度计和荧光显微镜检测△ψ;MTT比色法检测细胞活力;光镜观察细胞形态学变化;吖啶橙(AO)染色检测细胞凋亡.结果:与对照组相比,1 mmol/L H_2O_2处理的FRTL细胞10 min、30 min、24 h,细胞内MitoSOX荧光强度明显增强,rh123荧光强度和MTT吸光度明显下降(P＜0.01),光镜下可见细胞脱壁、破碎,AO染色可见核变小、变圆,染色质浓缩、边集,核碎裂改变.结论:1 mmol/L H_2O_2急性处理(10 min,30 min)和慢性处理(24 h)均能明显增加FRTL细胞线粒体超氧化物生成,降低线粒体膜电位,造成细胞坏死和凋亡.     

姜黄素和顺铂联用对雄激素非依赖性前列腺癌细胞PC-3作用

目的:研究姜黄素和顺铂联用抑制雄激素非依赖性前列腺癌细胞株PC-3增殖的影响.方法:MTT比色法检测细胞生长活性,流式细胞仪测定细胞周期时相变化及检测细胞凋亡,透射电镜观察细胞超微结构变化.结果:姜黄素能显著抑制雄激素非依赖性前列腺癌细胞的体外生长,呈时间与剂量依赖性;细胞周期主要阻滞于G2/M期,部分细胞出现凋亡形态学改变.顺铂和不同浓度姜黄素联合可显著抑制PC-3细胞增殖.姜黄素和顺铂在一定程度上均可诱导PC-3细胞凋亡,两种药物联用后,诱导细胞凋亡作用增强.结论:姜黄素可与顺铂协同抑制人前列腺癌细胞株PC-3的增殖.     

蛇床子素诱导小鼠成纤维细胞凋亡及其机制研究

目的:研究蛇床子素诱导小鼠胚胎成纤维细胞(NIH3T3)凋亡及其机制.方法:体外培养NIH3T3,不同浓度的蛇床子素作用于NIH3T3,应用溴化四氮唑蓝(MTT)比色法和生长曲线法检测蛇床子素对NIH3T3增殖活性的影响.Hoechst 33342荧光染色后,荧光显微镜下观察细胞核的变化.琼脂糖电泳分析法观察NIH3T3的DNA降解断裂的情况.结果:蛇床子素能明显抑制NIH3T3的生长,浓度为2.5×10-4mol/L时,细胞生长受到显著抑制,2.5×10-4mol/L蛇床子素作用24 h即可明显诱导NIH3T3的凋亡.结论:蛇床子素可明显抑制NIH3T3的生长并可以诱导其凋亡.     

四甲基偶氮唑蓝比色法测定促肝细胞生长素对SMMC-7721细胞的作用

目的:建立一种用四甲基偶氮唑蓝(MTT)比色法测定促肝细胞生长素对SMMC-7721细胞的促生长作用,以测定其生物活性的方法。方法:在体外采用MTT法,观察促肝细胞生长素在不同浓度(6.2~800.0mg.L-1)及不同的培养时间(24,48,72h),对不同浓度SMMC-7721细胞(1.5~100.0)×103个.mL-1生长的影响作用,并考察了此方法的重复性、与对人胃癌MGC-830及结肠癌HT-29细胞相比较的特异性及与法定3H-TdR掺法比较的相关性。结果:MTT法测定结果表明,浓度为100mg.L-1的促肝细胞生长素在培养时间为48h时,对细胞浓度为2.5×104个.mL-1的SMMC-7721细胞促生长作用最明显(刺激指数SI值≥6.0),此方法重复性好,并对SMMC-7721细胞具有特异性,与3H-TdR掺法比较测定的SI值差异无显著性。结论:MTT法适用于促肝细胞生长素的活性测定。     

改良MTT法检测HePG-2细胞增殖的研究

MTT比色法广泛用于检测培养细胞的生长和增殖、评价化学物的细胞毒性以及抗肿瘤药物的敏感试验。由于MTT的代谢产物甲瓒(formazan)为水不溶性的,必须采用合适的有机溶剂溶解后,才能通过酶标仪或分光光度计进行检测。通常的方法是在加入噻唑蓝(MTT)培养4 h后,吸出培养基,加入DMS     

迷迭香酸通过线粒体通路诱导骨肉瘤MG-63细胞凋亡的研究

目的研究迷迭香酸通过线粒体通路诱导骨肉瘤MG-63细胞凋亡的作用。方法体外培养MG-63细胞,将10、20、30、40、50、60μmol/L迷迭香酸作用于MG-63细胞,MTT和CCK-8法检测迷迭香酸对细胞生长的影响;AO/EB染色法观察细胞凋亡;流式细胞仪检测对细胞凋亡和细胞周期的影响;Western blotting法检测Cyt-c、Caspase-3、Bax和Bcl-2蛋白表达水平。结果 MTT和CCK-8结果显示迷迭香酸可浓度相关性地抑制MG-63细胞的生长;AO/BE染色可看到迷迭香酸组凋亡细胞明显增加;流式细胞仪检测结果显示迷迭香酸可浓度相关性地促进MG-63细胞凋亡,并将细胞周期阻滞在S期;Western blotting法结果显示与对照组比较,迷迭香酸20、40、60μmol/L组的Bax、Caspase-3和Cyt-c蛋白表达水平明显增加(P0.05、0.01);与对照组比较,迷迭香酸20、40、60μmol/L组的Bcl-2蛋白表达水平则明显降低(P0.05、0.01),且呈剂量相关性。结论迷迭香酸可显著促进人骨肉瘤MG-63细胞凋亡,其机制可能与调控线粒体凋亡通路有关。     

Limitation of the MTT and XTT assays for measuring cell viability due to superoxide formation induced by nano-scale TiO2

The reduction of the tetrazolium salts, MTT and XTT, is used to estimate cell viability and proliferation. However, superoxide can also reduce tetrazolium salts to produce the absorbant formazan end products. Evidence indicates that nano-TiO₂ induces superoxide formation in different mammalian cells. Therefore, studies investigating the cytological effects of nano-TiO₂ may encounter misleading results when using MTT/XTT to measure viability or proliferation. In this study, cell viabilities of Chinese hamster ovary cells were assayed using MTT, XTT and the trypan blue exclusion assay following exposure to nano-TiO₂. In comparison to the trypan blue exclusion assay, the MTT and XTT assays inaccurately predicted cell toxicity or overestimated cell viability respectively. XTT, in particular, appears more sensitive to superoxide than MTT. The reduction rate of XTT is 1.5 times that of MTT and SOD inhibition of XTT is less effective than that of MTT, indicating that XTT is more reactive with than MTT. Therefore, using XTT or MTT for measuring cell viability or proliferation may yield inaccurate results when conditions in cultured cell increase superoxide formation.     

The tetrazolium dye assay for rapid in vitro assessment of cytotoxicity

The intracellular reduction of a tetrazolium salt (MTT) to a purple formazan is an indicator of cell viability. The MTT test represents a simple colorimetric method to determine cytotoxicity. The present paper describes a new method for solubilisation of the formazan crystals, which makes the assay simpler, more rapid and more reproducible. Different cell lines show different kinetics of formazan formation stressing the need for individual calibration curves. The clear-cut discrimination between cells resistant and non-resistant against oxazaphosphorines indicates that the assay has predictive value for in vivo drug sensitivities. It is concluded that the MTT test is a useful addition to anticancer compound screening programs.     

In vitro bioassay for transforming growth factor-beta using XTT method

Research in the cytokine field has grown exponentially in recent years, and the validity of such studies relies heavily on the appropriate measurement of levels of cytokines in various biological samples. Transforming growth factor (TGF)-beta, a hormonally active polypeptide found in normal and transformed tissue, is a potent regulator of cell growth and differentiation. The most widely used bioassay for TGF-beta is the inhibition of the proliferation of mink lung epithelial cells. Though detection of [3H]thymidine incorporation is more sensitive than the MTT assay, it presents some disadvantages due to the safety and disposal problems associated with radioisotopes. In this study, we attempted to ascertain the experimental conditions which could be used for measuring the in vitro biological activity of TGF-beta in a safer and more sensitive way compared with the currently available methods. We compared the commonly used method, the MTT assay, to the XTT assay using different parameters including cell number, incubation time and the wave length used for detecting the product. We examined the anti-proliferative activities of TGF-beta in three different cell lines: Mv-1-Lu mink lung epithelial cells, MCF10A human breast epithelial cells and H-ras-transformed MCF10A cells. Herein, we present an experimental protocol which provides the most sensitive method of quantifying the biological activity of TGF-beta, with a detection limit of as low as 10 pg/ml: Mv-1-Lu or H-ras MCF10 A cells (1 x 10(5)/well) were incubated with TGF-beta at 37 degrees C in a humidified CO2 incubator for 24 hr followed by XTT treatment and determination of absorbance at 450 or 490 nm. Our results may contribute to the establishment of an in vitro bioassay system, which could be used for the satisfactory quantitation of TGF-beta.     

用细胞培养检测抗肿瘤药物敏感性研究中改良MTT光吸收技术与[~3H]-TdR掺入法的比较

应用改良四甲基偶氮唑盐(MTT)光吸收检测法,在体外肿瘤细胞培养中测定肿瘤化疗药物敏感性,并与[~3H]-TdR 掺入法进行平行比较实验。结果表明,改良 MTT 光吸收检测技术具有简便、快速、敏感性强的优点。本法无需接触同位素,安全,经济,便于实验室开展应用。     

吡柔比星和顺铂联合应用对人卵巢癌3AO细胞系的作用

目的：观察吡柔比星和顺铂联合应用对人卵巢癌3AO细胞系的抑制效应,并初步探讨其作用机制。方法：用MTT（四甲基偶氮唑盐）法检测化疗药物对人卵巢癌3AO细胞系的抑制率（Fa）;FCM（流氏细胞术）检测肿瘤细胞周期分布和细胞凋亡。结果：吡柔比星和顺铂单独用药可时间和浓度依赖性抑制3AO细胞的生长。IC50（半数抑制浓度）随时间延长而明显减小。联合应用吡柔比星和顺铂后抑制率呈时间和浓度依赖性。两药的IC50也比单独用药时显著降低;吡柔比星和顺铂舍用呈协同作用;两药单用和联合应用对细胞周期均有影响,细胞凋亡率均提高。结论：两药联合呈协同作用,药物的作用均与其诱导凋亡和影响细胞周期有关,但对细胞周期的影响各不相同。     

Comparison of XTT and Alamar blue assays in the assessment of the viability of various human cancer cell lines by AT-101 (−/− gossypol)

This study compared the two different commercially available in vitro viability assays: XTT and Alamar blue (AB), to detect anti-proliferative effects of AT-101, a cotton plant extract, on six different human carcinoma cell lines including: prostate (PC-3 and DU-145), breast (MCF-7 and MDA-MB-231), and ovary (OVCAR-3 and MDAH 2774) in a time- and dose-dependent manner. Cells were exposed to AT-101 in the concentration range of 2.5–40 µM for 24, 48, and 72?h. The AB assay was slightly more sensitive than the XTT assay in the evaluation of AT-101 at 24?h, suggesting that the AB assay might be used for detecting early changes in cell viability as compared to the XTT assay. Moreover, the AB assay showed less intra-assay variability as compared to the XTT. The non-toxic, non-radioactive AB metabolism assay allows rapid assessment of large numbers of samples, with simple equipment and at reduced cost for continuous monitoring of cancer cell viability, and, thus, should be accepted as a suitable alternative viability method.     

银杏内酯纳米粒细胞毒性研究

目的研究制备的银杏内酯纳米粒的细胞毒性作用。方法小鼠成纤维细胞L929在含银杏内酯纳米粒的培养液中培养7d,并与同浓度的银杏内酯药物和空白纳米粒(PBCA)比较,MTT法观测细胞形态变化,测量吸光度,计算细胞相对增值率。结果银杏内酯组、银杏内酯纳米粒组与空白纳米粒组对L929细胞的生长抑制作用无统计学差异(P>0.05),而且细胞毒性均为Ⅰ级。结论银杏内酯纳米粒对L929细胞无明显的细胞毒性。     

三氧化二砷通过JNK信号通路诱导K562细胞凋亡

目的:探讨c-Jun氨基末端激酶(JNK)信号通路在三氧化二砷(As2O3)诱导K562细胞凋亡中的作用及机制。方法:体外培养K562细胞,用As2O3及特异性JNK抑制剂SP600125对K562细胞进行处理;倒置相差显微镜下观察细胞形态学变化;MTT法检测不同时间点细胞增殖抑制率;AnnexinV/PI染色结合流式细胞术检测细胞凋亡率;ELISA检测p-JNK蛋白表达的变化;流式细胞术检测突变型P53表达。结果:ELISA显示4μmol/LAs2O3作用48h后p-JNK蛋白表达增强,经SP600125预处理后,As2O3诱导的K562细胞p-JNK蛋白表达明显减弱(P<0.01),As2O3诱导的细胞增殖抑制率和细胞凋亡率均下降,与As2O3单作用组相比突变型P53表达增加(P<0.05)。结论:JNK信号转导通路在As2O3诱导K562细胞凋亡过程中发挥重要作用,是As2O3诱导K562细胞凋亡的主要途径之一。     

A simple in vito cytotoxicity test using the MTT (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric assay: analysis of eugenol toxicity on dental pulp cells (RPC-C2A)

A simple colorimetric assay using MTT has been developed to monitor mammalian cell survival and proliferation in vitro. In this study we used a clonal fibroblastic cell line (RPC-C2A) from, rat incisal dental pulp to examine the effectiveness of the colorimetric assay to test for the toxicity of eugenol, which is frequently used to treat inflammed dental pulp. A technical problem encountered was the insolubility of MTT formazan, produced by the activity of mitochondria dehydrogenases. Dimethyl sulfoxide (DMSO) seemed to be the best solvent. Doses of eugenol causing a 50% inhibition in the colorimetric assay were calculated as 0.6 mM and 1 mM for cells in the growing phase and for cells at confluence, respectively. These values exist in the concentration range reported in the previous studies. Although the correlation between spectrophotometric absorbance and cell number was not completely linear, this method could be used effectively as a simple preliminary assay to test for the toxicity of dental drugs and materials.