流式细胞仪分选转基因斑马鱼胚胎荧光标记细胞的一种快速方法

荧光蛋白在特异组织和器官表达的转基因斑马鱼已经在发育生物学和疾病模型研究中得到了广泛的应用。这些转基因系有助于追踪和分析数量较少的细胞群。但是如果要分离得到这些细胞来定量分析mRNA或蛋白质的表达情况比较困难。利用流式细胞仪分选这些荧光标记细胞是一种解决办法。此方法在不同的实验室中被广泛地应用。也有相关流程的介绍。但是流程一般较为繁琐,操作比较困难。该文以从转基因斑马鱼Tg（Kdrl：EGFP）中分选绿色荧光蛋白阳性的血管内皮细胞为例,介绍利用流式细胞仪分选转基因斑马鱼荧光标记细胞的实验流程和技术要点。该文作者在前人工作的基础上结合大量的实验经验．发展并优化了一套操作简便、效率较高的流程来分选这些细胞,在此做详细的介绍给大家以供参考。     

利用流式细胞仪分选拟南芥根尖发育早期非根毛细胞

建立了应用流式细胞仪分选植物特定类型细胞的方法。以拟南芥（Arabidopsis thaliana）Wer：：GFP转基因株系为材料,用激光共聚焦显微镜鉴定GFP的表达位置,采用酶解法制备拟南芥根尖原生质体,应用流式细胞仪荧光激活细胞分选技术（FACS）分选收集GFP阳性细胞,并提取细胞的RNA。结果表明,Wer：：GFP转基因株系仅在根表皮发育早期的非根毛细胞中表达GFP;利用酶解法制备的根尖原生质体数目较多;从FACS分选收集的细胞中提取的RNA质量较好,可用于研究特定类型细胞的基因表达谱。应用流式细胞仪分选拟南芥非根毛细胞的方法为研究植物特定类型细胞的基因表达谱及基因功能奠定了技术基础。     

流式细胞仪分选人外周血T淋巴细胞的方法建立与评价

目的:利用流式细胞仪同时分离外人周血单个核细胞中T淋巴细胞并检测其分离纯度及存活率。方法:本文采用流式细胞仪同时分选人外周血CD4~+、CD8~+T淋巴细胞为例,推而广之,采用人外周血淋巴细胞分离液梯度离心法制备外周血单个核细胞,采用流式细胞仪同时分选CD4~+、CD8~+T淋巴细胞,分离细胞再通过流式细胞仪回测其分离纯度并通过台盼蓝染色检测分离细胞的存活率。结果:采用此方法能有效人外周血细胞CD4~+、CD8~+T淋巴细胞,分选前CD4~+淋巴细胞纯度为(50.5±11.5)%、CD8~+T淋巴细胞纯度为纯度为(15.4±7.1)%;分选后CD4~+T淋巴细胞纯度为(94.3±1.3)%、CD8~+T淋巴细胞纯度为(93.6±1.6)%;分选后CD4~+T淋巴细胞存活率为(95.3±1.8)%,CD8~+T淋巴细胞存活率为(94.8±1.5)%,细胞的形态完整。结论:采用人外周血淋巴细胞分离液梯度离心法制备外周血单个核细胞后利用流式细胞仪分选的方法能够高效、快速的分离人外周血CD4~+、CD8~+T淋巴细胞,且存活率高,为进一步研究其功能提供了保证。采用不同的荧光抗体标记其他淋巴细胞亚群,也能高效、快速的分离出细胞。     

一种应用流式细胞术快速分选活的衰老细胞的方法

衰老细胞是细胞老化研究的重要模型,目前还缺乏从混合细胞群中分选活的衰老细胞的有效方法。该研究以1.0μg/m L顺铂诱导的人鼻咽癌CNE 2细胞衰老为研究模型,根据细胞大小和自发荧光(脂褐素积累)两项物理特征,采用FACS Aria III流式细胞仪在Purity模式下设门分选活的衰老细胞(SEN)和增殖细胞(PROL),收集细胞,再分别进行回测分析和衰老相关β-半乳糖苷酶(SA-β-gal)染色验证。结果从SEN门控获得分选纯度为74.7%的活的衰老细胞,分选出的细胞可重新贴壁;其中,72.1%的细胞呈SA-β-gal染色阳性,与PROL门控分选获得的细胞(8.01%)相比较有显著差异(P0.01)。该研究提供了一种基于流式细胞仪快速分选活的衰老细胞的方法。     

捷安肽素高产突变株96孔板筛选方法的建立

建立了一种快速灵敏地筛选出捷安肽素(JAA)高产突变株的方法。主要利用96多孔板固体培养菌体,用50%乙醇为最佳浸提液浸提4h,滤纸片法检测JAA生物活性的点样量为100μL,筛选出5株高产突变株,经摇瓶复筛选出2株高产突变株,经一剂量法检测,其抑菌圈直径较出发菌株提高了14%左右。     

不同培养基对流式细胞仪分选外周血T细胞的影响

摘要 目的：通过探讨用于流式分选的T细胞体外扩增的无血清培养基，提高过继细胞的增殖能力和活性。方法：采用人外周血淋巴细胞分离管制备外周血单个核细胞，再用流式细胞分选仪从6例健康志愿者的外周血单个核细胞中分选CD3+T细胞到4种常用的培养基中：X-VIVO 15、KBM 581、TexMACS GMP和10 % FBS/1640，观察并记录培养细胞的状态和体外增殖能力。于第3天，第6天和第8天，通过胎盼蓝染色后进行活细胞计数。于第8天用凋亡试剂盒检测扩增细胞的凋亡情况，并用流式细胞分析仪检测细胞的免疫表型。结果：X-VIVO 15、TexMACS GMP和10 % FBS/1640作为流式细胞分选的接收液仅少量细胞碎片，而分选在KBM 581的细胞大量死亡，显著高于X-VIVO 15组（P<0.05）。X-VIVO 15中扩增的细胞数量最多，增殖检测结果显示活细胞在X-VIVO 15中快速增殖且细胞凋亡率显著低于KBM 581 和 TexMACS GMP（P<0.05）。4种培养基扩增的细胞主要呈现效应记忆型。其中，X-VIVO 15中效应记忆型T细胞比例显著高于TexMACS GMP（P<0.05）。TexMACS中效应细胞比例显著高于10 % FBS/1640（P<0.05）。结论：X-VIVO 15无血清培养基扩增流式分选的T细胞具有高增殖能力、细胞活性和记忆表型，适用于经流式分选后细胞的体外扩增。     

基于荧光激活细胞分选技术的超高通量酶活性筛选方法及其应用

基于荧光激活细胞分选(FACS)技术的超高通量酶活性筛选方法是新出现的一类高通量筛选技术.它利用流式细胞仪高灵敏度、高通量的特点,能以极高的速度(108/天)对大容量酶基因文库进行筛选.FACS筛选技术的出现突破了常规筛选方法低效、耗时、费力等瓶颈问题,极大地提升了人类对大容量基因文库的探索能力,因此在新酶基因筛选、酶活性检测、酶定向进化等领域有广泛的应用潜力.综述了FACS超高通量酶活性筛选方法的最新研究进展,着重介绍了其在酶定向进化中的应用.     

应用两种方法诱导SH-SY5Y细胞分化的研究

目的：观察全反式维甲酸（ATRA）处理和ATRA与十四烷酰佛波醇乙酸酯（TPA）序贯处理（ATRA/TPA）对人类神经母细胞瘤细胞系SH-SY5Y细胞增殖抑制和形态分化的影响。方法：应用10μM ATRA处理6天和10μM ATRA处理3天继以80 nMTPA处理3天这两种方法使SH-SY5Y细胞分化;用倒置光学显微镜动态观察SH-SY5Y细胞形态学变化;并用MTT比色法比较两种分化方法对SH-SY5Y细胞的体外抗增殖作用。结果：ATRA处理和ATRA与TPA序贯处理对SH-SY5Y细胞都有抗增值和诱导细胞分化作用,细胞形态发生明显的变化,分化成神经元表型,前者主要表现为两端带有长突起的纺锤体样细胞形态,而后者主要是由细胞体延伸出多个突起的多边形的细胞。ATRA分化6天的细胞的存活率下降为78.7%±2.0%。当去除ATRA后,继续培养1天的细胞存活率上升为89%±0.2%,而继续培养2天的细胞存活率为86.3%±1.4%;ATRA与TPA序贯分化6天细胞存活率下降为75.9±0.4%。当去除TPA后,继续培养一天的细胞存活率为75.5±0.7%,继续培养2天的细胞存活率为74.9±1.0%。结论：维甲酸（ATRA）处理和ATRA与十四烷酰佛波醇乙酸酯（TPA）序贯处理（ATRA/TPA）均能明显诱导SH-SY5Y细胞分化。这两种分化细胞为神经科学的研究提供了优良的体外培养模型细胞,尤其是ATRA与TPA序贯处理能获得分化完全而稳定的神经元样细胞。     

A semiautomated 96-well plate assay for protein kinase C

We have devised a rapid procedure for the assay of protein kinase C. Reactions (100 microliters) were set up in the wells of a 96-well assay plate and initiated one column at a time by the addition of [gamma-32P]ATP with an eight-channel pipettor. After incubation for 5 min at 30 degrees C, the reactions were terminated by the addition of 100 microliters of 25% trichloroacetic acid. The reaction mixtures were then filtered using a semiautomatic cell harvester, and transferred to scintillation vials using a filter punch apparatus. Direct comparison of this method to traditional techniques revealed a three- to fivefold increase in efficiency with equal sensitivity. This method is suitable for screening large numbers of inhibitors and activators of protein kinase C and appears to be applicable to other enzymes such as calmodulin kinases.     

A nonradioactive 96-well plate assay for screening of trans-sialidase activity

Trans-sialidase (E.C. 3.2.1.18) catalyzes the transfer of preferably alpha2,3-linked sialic acid to another glycan or glycoconjugate, forming a new alpha2,3 linkage to galactose or N-acetylgalactosamine. Here, we describe a nonradioactive 96-well plate fluorescence test for monitoring trans-sialidase activity with high sensitivity, specificity, and reproducibility using sialyllactose and 4-methylumbelliferyl-beta-D-galactoside as donor and acceptor substrates, respectively. The assay conditions were optimized using the trans-sialidase from Trypanosoma congolense and its general applicability was confirmed with recombinant trans-sialidase from Trypanosoma cruzi. Using this procedure, a large number of samples can be tested quickly and reliably, for instance in monitoring trans-sialidase during enzyme purification and the production of monoclonal antibodies, for enzyme characterization, and for identifying potential substrates and inhibitors. The trans-sialidase assay reported here was capable of detecting trans-sialidase activity in the low-mU range and may be a valuable tool in the search for further trans-sialidases in various biological systems.     

A semiautomated, 96-well plate assay for collagen synthesis.

We have established a rapid method for the measurement of collagen synthesis in large numbers of cell cultures. 3H-labeled collagen in microwell cultures was salt precipitated, harvested, and washed using a commercially available cell harvester and the filtered collagen was directly counted. The cell number could then be assessed either by methylene blue staining or by dissolving the cells in NaOH and estimating the protein content with bicinchoninic acid. In all of these procedures, the samples remain in microtiter plates, thus ensuring that minimal amounts of reagents are used and minimizing the amount of manipulation necessary. The intergroup variability is 3 to 9% and the recovery of 3H-labeled collagen is greater than 90%. Using this method, large numbers of samples can be assessed for collagen synthesis quickly, conveniently, and for minimal cost.     

A continuous 96-well plate spectrophotometric assay for branched-chain amino acid aminotransferases

A new, continuous 96-well plate spectrophotometric assay for the branched-chain amino acid aminotransferases is described. Transamination of L-leucine with alpha-ketoglutarate results in formation of alpha-ketoisocaproate, which is reductively aminated back to L-leucine by leucine dehydrogenase in the presence of ammonia and NADH. The disappearance of absorbance at 340 nm due to NADH oxidation is measured continuously. The specific activities obtained by this procedure for the highly purified human mitochondrial and cytosolic isoforms of BCAT compare favorably with those obtained by a commonly used radiochemical procedure, which measures transamination between alpha-ketoiso[1-14C]valerate and L-isoleucine. Due to the presence of glutamate dehydrogenase substrates (alpha-ketoglutarate, ammonia, and NADH) and L-leucine (an activator of glutamate dehydrogenase) in the standard assay mixture, interference with the measurement of BCAT activity in tissue homogenates by glutamate dehydrogenase is observed. However, by limiting the amount of ammonia and including the inhibitor GTP in the assay mixture, the interference from the glutamate dehydrogenase reaction is minimized. By comparing the rate of loss of absorbance at 340 nm in the modified spectrophotometric assay mixture containing leucine dehydrogenase to that obtained in the modified spectrophotometric assay mixture lacking leucine dehydrogenase, it is possible to measure BCAT activity in microliter amounts of rat tissue homogenates. The specific activities of BCAT in homogenates of selected rat tissues obtained by this method are comparable to those obtained previously by the radiochemical procedure.     

Potency assays for Anistreplase: comparison of the fibrin plate assay and a 96-well plate assay.

Several production lots of Anistreplase (Eminase) were assayed for potency by either two fibrin plate assays or a clot lysis assay performed in 96-well microtiter plates. The 96-well plate assay yielded comparable data to the fibrin plate assays and had the advantage of greater efficiency with respect to both time and reagents. As a result the newer method appears to be a suitable alternative to the fibrin plate assays for lot release of Anistreplase.     

A colorimetric 96-well microtiter plate assay for the determination of enzymatically formed citrulline

l-Citrulline constitutes a product of a number of enzymatic reactions. In the past a number of colorimetric methods for the determination of l-citrulline, upon its chemical modification with diacetyl monoxime at 95 degrees C, have been reported. However, all these methods are time- and material-consuming. In this work, using the same chemical reaction, a new method for the use in 96-well polystyrene microtiter plates was developed. The method is fast and requires substantially less material as the enzymatic reaction is performed in a volume of 60 microl. The applicability of this enzymatic assay was established using l-N(omega), N(omega)-dimethylarginine dimethylaminohydrolase, which generates l-citrulline from side-chain methylated derivatives of l-arginine. The detection limit for l-citrulline is about 0.2 nmol. In addition, our studies show that most commonly used biochemical buffers and buffer additives do not affect the assay. This method may prove useful in the studies of other l-citrulline producing enzymes including nitric oxide synthase.     

Measurement of hydrocarbon-degrading microbial populations by a 96-well plate most-probable-number procedure

A 96-well microtiter plate most-probable-number (MPN) procedure was developed to enumerate hydrocarbondegrading microorganisms. The performance of this method, which uses number 2 fuel oil (F2) as the selective growth substrate and reduction of iodonitrotetrazolium violet (INT) to detect positive wells, was evaluated by comparison with an established 24-well microtiter plate MPN procedure (the Sheen Screen), which uses weathered North Slope crude oil as the selective substrate and detects positive wells by emulsification or dispersion of the oil. Both procedures gave similar estimates of the hydrocarbon-degrader population densities in several oil-degrading enrichment cultures and sand samples from a variety of coastal sites. Although several oils were effective substrates for the 96-well procedure, the combination of F2 with INT was best, because the color change associated with INT reduction was more easily detected in the small wells than was disruption of the crude oil slick. The method's accuracy was evaluated by comparing hydrocarbon-degrader MPNs with heterotrophic plate counts for several pure and mixed cultures. For some organisms, it seems likely that a single cell cannot initiate sufficient growth to produce a positive result. Thus, this and other hydrocarbon-degrader MPN procedures might underestimate the hydrocarbon-degrading population, even for culturable organisms.     

Continuous spectrophotometric assay amenable to 96-well plate format for prostaglandin E synthase activity

The measurement of prostaglandin E synthase (PGES) activity is cumbersome because the product of the reaction, PGE(2), is not readily quantitated by spectral means. The activity of isolated PGES is typically determined by PGE(2) immunoassay or by high-performance liquid chromatography using radiolabeled substrate. A relatively rapid continuous spectrophotometric assay which uses 15-hydroxyprostaglandin dehydrogenase (PGDH) to couple the oxidation of the 15-hydroxy group of PGE(2) to the formation of NADH was developed. PGDH is relatively specific for PGE(2) over the substrate for the PGES reaction, PGH(2), allowing a highly reproducible assay of PGES activity to be obtained.     

Resazurin-based 96-well plate microdilution method for the determination of minimum inhibitory concentration of biosurfactants

Objectives

To develop and validate a microdilution method for measuring the minimum inhibitory concentration (MIC) of biosurfactants.

Results

A standardized microdilution method including resazurin dye has been developed for measuring the MIC of biosurfactants and its validity was established through the replication of tetracycline and gentamicin MIC determination with standard bacterial strains.

Conclusion

This new method allows the generation of accurate MIC measurements, whilst overcoming critical issues related to colour and solubility which may interfere with growth measurements for many types of biosurfactant extracts.