两种新型培养细胞数量检测技术的探索

将SRB比色法和结晶紫比色法与MTT比色法与MTT比色法在96孔板培养HeLa细胞的数量及活力测量精确度和操作方法上进行了对比。试验结果显示：SRB比色法和结晶紫比色法的最佳检测波长均为490nm。最佳染液浓度分别为0.4%和0.25%。SRB比色法的检测精确度略逊于MTT法，结晶紫比色法与MTT法无显著差异。另外，此两种检测方法的操作过程更为简便，所需时间也较短，可在一定范围内取代MTT比色法。     

两种体外细胞毒性检测方法的比较研究

目的比较两种常用的细胞毒性检测方法在医疗器械生物学评价中的相关性。方法分别采用MTT比色法和细胞增殖度法,在37℃条件下,将五种医疗器械／生物材料的浸提液分别与小鼠成纤维细胞（L-929）接触2天和2,4,7天,比较材料对细胞的毒性影响。结果5种不同的材料浸提液分别表现出不同程度的细胞毒性反应（0～2级）。将MTT比色法与细胞增殖度法（2天）的实验数据进行相关性分析,显示两者之间具有良好的相关性（R=0．977）。结论MTT比色法由于其检测所需的细胞量相对较少,试验步骤相对简便、检测周期短,因此具有一定的优越性,是个值得推荐的细胞毒性检测方法。     

地塞米松诱导培养的大鼠大脑皮质星形胶质细胞凋亡

目的　研究地塞米松诱导纯化培养的大鼠大脑皮质星形胶质细胞凋亡的作用。方法　不同浓度的地塞米松(浓度为 10 -3 、 10 -4、 10 -5mol/L)与纯化培养的大鼠大脑皮质星形胶质细胞共同孵育 18小时后 ,吖啶橙染色荧光显微镜观察细胞凋亡形态学改变 ,流式细胞仪检测细胞凋亡和结晶紫比色法酶标仪测定活细胞数。结果　 (1)吖啶橙染色荧光显微镜观察 :10 -4组偶见细胞凋亡 ,10 -3 组可见许多细胞有典型的凋亡形态学改变核固缩 ,深染 ,或肿胀 ,碎裂 ,并可见凋亡小体。 (2 )流式细胞仪检测细胞凋亡 :10 -3 组细胞凋亡率为 15 99% ,与其它三组相比明显增高 ,有显著性差异 (P <0 0 1)。 (3)结晶紫法酶标仪测定活细胞数 :10 -3 组OD值为 0 . 185与其它三组相比明显下降 ,有显著性差异 (P <0 0 1) ,表明活细胞数明显减少。结论　大剂量地塞米松可诱导体外的星形胶质细胞凋亡。     

用荧光激活细胞分选仪检测细胞存活的两种不同染色方法的比较

本文比较了用PI和FDA两种不同染料, 通过FACS来检测细胞的存活,表明两种不同染料所得结果均能正确反映细胞存活的情况,而且两者的结果是一致和稳定的。并讨论了这两种方法应用于FACS检测中的优点。     

MTT比色法抗肿瘤药物筛选实验条件和数据优化探索

MTT比色法是一种重要的体外抗肿瘤药物筛选方法.以PC-3细胞系为研究对象,对影响MTT比色法抗肿瘤药物筛选实验的主要因素一细胞密度、实验操作环节、OD值选取、以及数据的优化处理进行实验探讨.结果表明,当检测化合物6 h～72 h的抑制活性时,种细胞密度2 000个/孔为宜.同时,采用本文所提的实验条件和数据处理办法,可以实现实验结果的准确可靠,3次测试偏差不超过10%.     

一种快速的活菌计数新方法研究*

四唑盐（MTT）比色法是一种检测动物细胞活细胞数的方法。通过改变比色反应温度、反应时间和比色波长,建立了一种快速、准确的活菌计数新方法并将其应用于细菌PBW1培养过程中活菌浓度的测定。结果表明,新方法与平板稀释法的测定结果一致,且具有快速、方便等优点。     

一种快速的活菌计数新方法研究

四唑盐（MTT）比色法是一种检测动物细胞活细胞数的方法。通过改变比色反应温度、反应时间和比色波长,建立了一种快速、准确的活菌计数新方法并将其应用于细菌PBW1培养过程中活菌浓度的测定。结果表明,新方法与平板稀释法的测定结果一致,且具有快速 、方便等优点。     

微孔比色法在猪胰腺PLA2制备中的应用

微孔比色法采用合成的磷脂类似物２－硫代十六酰乙基磷酸胆碱作底物,在多孔聚苯乙烯板的小孔中反应,并用酶联免疫检测器连续测定和记录吸收值。同时应用此法及滴定法检测酶活力,从猪胰腺中制备了一种分子量低（１４．３ｋＤ）,对热、酸稳定,活性依赖Ｃａ＾２＋的ＰＬＡ２。两种方法检测结果具有可比性,而微孔比色法同时可测多个样品,有节约样品,灵敏度较高等优点。微孔比色法特别适用于大量的样品测定,如拮抗剂筛选、临床样     

视网膜神经节细胞的纯化和体外存活

我们用特异性抗体Thy1.1结合尼龙筛方法分离和纯化新生大鼠视网膜神经节细胞,比较顶盖提取液对这些纯化细胞的作用。预先以快蓝(fast blue,FB)逆行标记的视网膜细胞悬液,接种在包被了Thy1.1抗体的培养皿上30分钟,冲洗未粘附的细胞,显微镜下计数粘附细胞中FB标记的视网膜神经节细胞纯度的百分比,最高为95%。用孔径15μm尼龙筛方法分离的纯度仅为60±5%。上述两种方法纯化的视网膜神经节细胞,仅在有顶盖提取液存在时,细胞存活并生长活跃,胞体大且有突起伸出。MTT微量比色法测定培养24小时纯化细胞存活的光密度(OD)值,显示以Thy1.1特异性抗体纯化的细胞,其OD值比值(+Te/-Te)是12.3(0.111/0.009);以尼龙筛纯化的OD值比值(+Te/-Te)是6.4(0.102/0.016);未经纯化的OD值比值(+Te/-Te)是3.8(0.095/0.025)。在上述三组中,加Te与无Te细胞生存的OD值比较,相差均非常显著(P<0.01)。结论:在纯化的视网膜神经节细胞的培养中,由于排除了其他细胞所引起的非特异性反应,神经节细胞能够更直接地反映顶盖提取液的生物效应;视网膜神经节细胞纯度越高,其作用越显著。     

严重发热伴血小板减少综合征病毒培养以及病毒滴度检测方法的建立

目的:建立严重发热伴血小板综合征病毒(SFTSV)培养和病毒滴度检测的方法,为其致病机制研究奠定基础。方法:选取生长良好的Vero细胞铺六孔板接种SFTSV,每隔24 h收集培养病毒上清检测SFTSV病毒复制拷贝数,从而选取最佳时间点收获病毒液并用浓缩离心管浓缩病毒液,保存备用。将SFTSV病毒液进行10倍比稀释,每梯度200μL接种生长良好的单层Vero细胞,用3 m L高压灭菌的甲基纤维素-DMEM覆盖物覆盖每孔Vero细胞,约9 d后温和去除甲基纤维素-DMEM覆盖物,经预冷的4%甲醛固定细胞后,用结晶紫染色,PBS洗脱3次,计算空斑数。结果:根据病毒繁殖生长情况,病毒在第4 d后达复制高峰并随后进入平台期,第8天病毒复制开始下降。参照甲基纤维素-结晶紫空斑法实验结果,病毒滴度为6×106PFU/m L。结论:SFTSV病毒培养以及滴度检测方法成功建立,选取4天后收获病毒上清最佳,甲基纤维素-结晶紫空斑法形成的空斑清晰可数。     

MTT比色法测定促肝细胞生长物质对肝细胞生长的刺激活性

本实验建立了用简便的ＭＴＴ比色法对促肝细胞生长物质的促肝细胞增殖作用的测定方法,确定了实验的最适条件。与传统的３Ｈ ＴｄＲ掺入法进行比较的结果显示,ＭＴＴ比色法与３Ｈ ＴｄＲ掺入法测定结果基本相符,灵敏度相近,但消除了同位素的污染,是一个测定促肝细胞生长物质刺激肝细胞增殖活性的简便方法。     

木豆叶总黄酮测定方法的比较研究

采用比色法测定木豆叶中的总黄酮含量。对三氯化铝比色法、硼酸—柠檬酸比色法和硝酸铝比色法3种测定方法进行比较,确定了硝酸铝比色法为木豆叶总黄酮的最佳测定方法,该方法稳定性、精密度和重现性好,其RSD分别为2.1%、0.9%和2.3%,应用该方法测定木豆叶总黄酮含量为15.65 mg·g^-1 DW。本方法适用于木豆叶或其制剂中总黄酮的质量分析检验,为木豆叶中黄酮的研究开发提供了质控依据。     

Application of Colorimetric Assays to Assess Viability, Growth and Metabolism of Hydrogel-Encapsulated Cells

The applicability of the colorimetric 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays to measure cell growth and viability in hydrogel encapsulation systems was investigated using HepG2 liver cells encapsulated in alginate matrices. The MTT assay was effective in measuring viable cell density in alginate-encapsulated cell systems, demonstrating less variance and higher throughput capability than hemocytometry. The LDH assay was effective in measuring dead cell density in monolayer cultures and in alginate-encapsulated cells simply by measuring the LDH concentration secreted into the medium. Further validation of these assays was shown in two additional cell lines (rat muscle and mouse embryonic fibroblasts). The MTT and LDH assays are particularly significant in the rapid evaluation of in vitro cell encapsulation device design.     

An assay for functional dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) inhibitors of human dendritic cell adhesion

We report a new dendritic cell adhesion assay, using either immature or mature dendritic cells, for identifying functional dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) inhibitors. Because immature dendritic cells are responsible for pathogen binding and invasion, this in vitro assay provides an important link between in vitro results and pathogen-based in vivo assays. Furthermore, this assay does not require laborious expression, refolding, and purification of DC-SIGN carbohydrate recognition domain or extracellular domain as receptor-based assays. The assay power evaluated with Z and Z′ parameters enables screening of compound libraries and determination of IC₅₀ values in the first stage of DC-SIGN inhibitor development.     

SRB法和MTT法抗肿瘤药物筛选结果相关性研究

为了探索抗肿瘤药物体外筛选中磺酰罗丹明B（SRB）法和四甲基偶氮唑盐（MTT）比色法实验结果的相互验证作用,作者以PC-3、BGC-823、Bcap-37细胞系为研究对象,利用SRB法和MTT比色法对供试化合物行体外抑制活性考察,并用SPSS12．0对两种筛选方法得到的实验结果进行差异性分析。结果表明,SRB法和MTT比色法实验结果相关性好,可以相互验证实验结果的准确性和可靠性。     

抗肿瘤药物筛选中MTT法和SRB法的比较

在抗肿瘤药物的体外筛选中 ,MTT法和 SRB法是常用的两种方法。我们用MTT法和 SRB法分别测定 3种已知植物抗癌药对 2 2株人肿瘤细胞的抗癌活性 ,对这两种方法进行了详细的比较。通过分析两种方法测出的细胞存活率 ( T/ C)的差异分布和相关系数以及 IC50 的二变量分布 ,比较了两种方法测定结果的异同 ;通过两种方法重复测定 3种药物对 7株人癌细胞的抗癌活性 ,比较了两种方法的重复性 ;通过分析两种方法测定结果 T/ C值随时间变化的程度 ,比较了两种方法测定结果的稳定性。实验结果表明 :MTT法和 SRB法的相关性较好 ,都可用于抗肿瘤药物的体外筛选 ,SRB法更适合于大规模筛选 ,3种抗癌药物的测定结果与临床资料基本一致。     

Assessing the data quality in predictive toxicology using a panel of cell lines and cytotoxicity assays

In vitro cell viability assays have a central role in predictive toxicology, both in assessing acute toxicity of chemicals and as a source of experimental data for in silico methods. However, the quality of in vitro toxicity databanks fluctuates dramatically because information they contain is obtained under varying conditions and in different laboratories. The aim of this study was to identify the factors responsible for these deviations and thus the quality of the data extracted for predictive toxicology. Three cell viability assays measuring LDH leakage, WST-1 reduction, and intracellular ATP were compared in an automated environment using four mammalian cell lines: Caco-2, Calu-3, Huh-7, and BHK. Using four standard compounds--polymyxin B, gramicidin, 5-fluorouracil, and camptothecin--a significant lack of sensitivity in LDH assay compared with the other assays was observed. Because the viability IC(50) values for the standards were similar among the cell lines, the biochemical characteristics of different cell lines seem to play only a minor role, with an exception being the hepatocellular Huh-7 cell line. Toxicity assessment of new 1,2,4-triazoles revealed significant differences in their toxic potential, and the results indicate the same sensitivity profile among the assays as observed with the standard compounds. Overall, it can be argued that the assay selection is the most important factor governing the uniform quality of the data obtained from in vitro cell viability assays.     

Apoptosis enzyme-linked immunosorbent assay distinguishes anticancer drugs from toxic chemicals and predicts drug synergism

The effects of anticancer drugs and toxic compounds on leukemic cells in culture were evaluated by enzyme-linked-immunosorbent assay (ELISA) based on the detection of apoptotic cells by a monoclonal antibody against single-stranded DNA. The concentrations of 13 anticancer drugs, which increased apoptosis ELISA absorbance, were similar to the concentrations decreasing long-term cell survival. Short-term metabolic tetrazolium-based 3-(4,5-dimethylthiazol-yl)-2,5-diphenyformazan bromide (MTT) assay was significantly less sensitive than apoptosis ELISA and the cell survival assay. In contrast to anticancer drugs, 12 toxic chemicals did not increase apoptosis ELISA absorbance at cytotoxic concentrations. The difference between two groups of compounds by apoptosis ELISA was especially large in cultures treated with twofold of concentrations producing 50% inhibition of cell growth: all anticancer drugs induced intense reaction (mean absorbance 2.0), while none of the toxic chemicals induced apoptosis. The application of apoptosis ELISA to chemosensitivity testing was evaluated by its ability to detect synergism of anticancer drug combinations. Among 66 drug combinations tested, only combination of nitrogen mustard with mithramycin was highly synergistic by the apoptosis ELISA, as defined by apoptosis induction with the combination containing each drug at 50% of effective concentration. This combination was also synergistic in the cell survival assay, producing significant cell kill while each drug alone had no effect on cell survival. This synergism was not detected by MTT assay. We conclude that apoptosis ELISA could be useful for drug development and chemosensitivity assessment as it can distinguish clinically useful anticancer drugs from toxic compounds, is as sensitive as the long-term cell survival assay and can detect anticancer drug synergism by rapid evaluation of apoptosis induction.     

A rapid micro method for counting cells “in situ” using a fluorogenic alkaline phosphatase enzyme assay

Summary A new method has been developed to count cells “in situ”, based on a fluorogenic enzyme assay that measures the activity of alkaline phosphatase. Increasing cell number was shown to correlate closely with alkaline phosphatase activity and this relationship did not change with time in culture. The alkaline phosphatase assay (ALP assay) was able to estimate relative cell numbers over a range from about 10⁴ to 5×10⁵ for many cell types, including Hep-2, a derivative of HeLa, several human colorectal cell lines SW1222, SW837, LS174T and HT29, a normal human diploid cell strain MRC5 and a rodent line NIH-3T3. The ALP assay is rapid and efficient, making it a useful method for studying growth assays. Editor's Statement This paper describes a quick method for quantitation of cell number in microcultures. Such procedures are valuable for the many situations in which minimizing cells and medium volume is desirable, although somewhat specialized equipment is required for the procedure. An alternative procedure for quantitation of cells in microtiter culture appeared previously in this journal (McCaffrey, et al., 24∶247–252).     

Microplate‐reader compatible perfusion microbioreactor array for modular tissue culture and cytotoxicity assays

One important application of tissue engineering is to provide novel in vitro models for cell‐based assays. Perfusion microbioreactor array provides a useful tool for microscale tissue culture in parallel. However, high‐throughput data generation has been a challenge. In this study, a 4 × 4 array of perfusion microbioreactors was developed for plate‐reader compatible, time‐series quantification of cell proliferation, and cytotoxicity assays. The device was built through multilayer soft lithography. Low‐cost nonwoven polyethylene terephthalate fibrous matrices were integrated as modular tissue culture scaffolds. Human colon cancer HT‐29 cells with stable expression of enhanced green fluorescent protein were cultured in the device with continuous perfusion and reached a cell density over 5 × 10⁷ cells/mL. The microbioreactor array was used to test a chemotherapeutic drug 5‐FU for its effect on HT‐29 cells in continuous perfusion 3D culture. Compared with conventional 2D cytotoxicity assay, significant drug resistance was observed in the 3D perfusion culture. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010