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。     

细胞计数板在荧光显微镜观察B细胞吞噬现象的应用探讨

目的:基于细胞计数板建立一种简单、快速使用免疫荧光显微镜观察B淋巴细胞吞噬卡介苗(BCG)现象的新方法,对即将进行流式细胞检测的样品进行质控,提高流式细胞术检测吞噬率的稳定性,同时为流式细胞仪检测吞噬率提供镜下依据。方法:B细胞与FITC标记的BCG共培养24 h后,PE anti-human CD19抗体直接标记细胞膜,应用细胞计数板在荧光显微镜下观察B细胞吞噬现象,流式细胞仪检测吞噬率。结果:应用细胞计数板在荧光镜下可观察到B细胞与BCG的荧光标记及B细胞与BCG共标记现象,证实B细胞可吞噬BCG,流式细胞仪检测结果显示吞噬率为13.9%。结论:应用细胞计数板在荧光镜下可观察B细胞吞噬现象,且操作简便快速,能对流式细胞检测的样品进行质控,并提供镜下依据。     

流式细胞术

流式细胞术是一种综合应用光学、机械学、流体力学、电子计算机、细胞生物学、分子免疫学等学科技术,对高速流动的细胞或亚细胞进行快速定量测定和分析的方法。它一秒钟能分析几千个细胞,并同时测定细胞的多个参数,广泛应用于生物医学的许多领域,如测定细胞的特征(形态、膜电位等)和细胞内pH,细胞DNA、蛋白质含量、表面受体、Ca2+等。对生物工程学来说,了解细胞的这些参数尤为重要,因为它们能比用传统技术测得的数据更好地描述细胞群体。从流式细胞仪对细胞多种参数的测定及原理,到它在生物工程学中的应用等方面进行了介绍,并讨论了流式细胞术的局限性和面临的挑战。     

专家答读者问：流式细胞仪如何将不同类型的细胞分开

<正>流式细胞仪（flow cytometer）是一种在功能水平上对单细胞或其他生物粒子进行定量分析和分选的检测仪器，可以每秒钟分析上万个细胞/粒子，并能同时从一个细胞/粒子中获得多个参数。其应用范围非常广泛，而且还在不断拓展，细胞分选也是它的重要应用之一。它能够根据每个细胞的光散射和荧光特征，将特定的细胞从细胞群体中分选出来。下文以细胞为例具体说明流式细胞仪的工作原理、技术特点和应用。流式细胞仪能够利用细胞的光散射特征，     

流式细胞术在高等植物研究中的应用

流式细胞术(FCM)是根据所测定的各种细胞性质的不同组合,从细胞群体中把某个亚群分选出来,并对它的功能和形态学进行研究或进一步培养分析。流式细胞术具有快速、灵敏和同时进行多参数检测等优点,对其基本原理和在高等植物中的应用进行了介绍。     

流式细胞仪使用中的一些技术问题

流式细胞仪(Flow Cytometer,FCM)集近代激光、光学、流体力学、电子计算机和精密机械等发展成果于一身,是目前细胞研究工具中最精密、最先进的仪器之一,国外科研和医疗单位已广泛使用,国内正在引入、试制,逐步普及。     

微核技术研究进展

微核试验作为检测染色体损伤最常用而有效的细胞遗传学检测方法之一,经济、简便、快速,在敏感性、特异性和准确性方面,与经典的染色体畸变分析方法基本相当。主要综述国内外微核检测技术的最新研究进展,尤其是微核的自动化检测技术。其中流式细胞仪自动化检测和激光扫描细胞仪自动化检测,以及微核试验高内涵筛选方法由于其特有的优势,应用和发展前景广阔。     

流式细胞仪的原理、应用及最新进展

流式细胞术是一种采用激光束激发单行流动的细胞,对它的散射光和携带的荧光进行探测,从而完成细胞分析和分选的技术。以流式细胞术为核心技术,流式细胞仪集光学、电子学、生物学、免疫学等多门学科和技术于一体,能够高效分析微小颗粒(如细胞,细菌)的先进科技设备。它对社会产生了深远的影响,成为了科学研究的必要工具。最近几年,流式细胞仪取得了长足进步。为了深入的了解它,本文从流式细胞仪的工作原理和技术指标,在临床医学、生物学、生殖学和制药学中的应用,以及它的世界格局、仪器功能的最新进展三方面,进行了简明、扼要的论述。展望未来:功能专业化、自动化,体积小型化,多色多参数分析能力提高和分析分选速度更快成为流式细胞仪发展的趋势。     

流式细胞技术在抗生素敏感性试验中的应用

流式细胞技术(FCM)是利用流式细胞仪对生物颗粒的多种物理和生物学特性进行定量分析.流式细胞荧光技术药敏试验(FCST)则是根据FCM所检测到的荧光强度来判断经药物处理后的培养液中细菌或真菌的存活率,本文综述这一技术的应用.     

流式细胞术检测组蛋白乙酰化水平方法的建立与应用

组蛋白去乙酰化酶抑制剂是近年来出现的一类新的抗肿瘤药物,在艾滋病等其他疾病中同样也受到关注。但是在基础和临床研究中,目前还缺乏统一可靠的组蛋白乙酰化水平的检测手段。本文利用全血和外周血单个核细胞,通过一系列的对比实验,比较了不同样品处理温度(冰上和室温)、破膜方法(细胞内因子染色破膜和核内因子染色破膜)、抗体剂量(抗体滴定)和抗体孵育时间(时间梯度)等实验条件对流式细胞术检测的影响,最终建立了一套基于流式细胞术的组蛋白乙酰化水平检测手段。同时,将优化后的流式细胞检测技术应用于西达本胺(目前国内唯一上市的组蛋白去乙酰化酶抑制剂)的体外实验和临床试验,结果均证明本文建立的组蛋白乙酰化流式细胞检测方法可以作为基础和临床研究中一个可靠、快速、便捷的检测手段。     

Enumeration of small ciliates in culture by flow cytometry and nucleic acid staining

We developed a fast and simple protocol for accurate quantification of small freshwater ciliates by flow cytometry (FCM). The ciliates were stained with several nucleic acid stains such as TO-PRO-1, YO-YO-1 and PicoGreen, and analysed by a commercially available flow cytometer. The method was tested with cultures of the prostomatid species Urotricha farcta and Balanion planctonicum, including the small cryptophyte Cryptomonas sp. as food. Of the dyes tested, TO-PRO-1 gave the best results. Flow cytometric results agreed well with microscopic counts. Due to its greater speed and accuracy, FCM was superior to light microscopy. FCM was also superior to electronical particle counting and sizing (EPCS). Of particular importance, FCM in combination with TO-PRO-1 staining allowed unequivocal discrimination in cases of overlapping size distributions between the target population (i.e., the ciliate predators) and other particles (the cryptophyte prey, detritus).     

Programming for the DNA analysis of FCM data on an IBM microcomputer

The analysis of data generated on a flow cytometer (FCM) is often performed on a computer obtained especially for dedicated use with the flow cytometer. This computer component can be expensive and also presents the FCM user with the added burden of mastering specialized programming language or of accepting the secret analytical processes of protected proprietary program routines. We believe that the evolution of more accurate and efficient FCM analyses that have the power to consider complex signal distributions can be assisted by the availability of analysis programs written in languages common to many users. DNA analysis routines written for a relatively inexpensive microcomputer (IBM PC/XT) in Basic and Pascal are described here. The routines can automatically process multiple FCM data files and can provide high-resolution graphic hardcopy. A foreground/background utilization is also described that allows the computer to be available for other uses in the laboratory.     

Bacterial fingerprinting by flow cytometry: bacterial species discrimination.

BACKGROUND: A flow cytometric measurement (FCM) technique has been developed to size DNA fragments. Individual fragments of a restriction digest of genomic DNA, stained with an intercalating dye, are passed through an ultrasensitive cytometer. The measured fluorescence intensity from each fragment is proportional to the fragment length. METHODS: The isolation of bacterial genomic DNA and digestion by restriction enzymes were performed inside an agarose plug. Rare cutting enzymes were employed to produce a manageable number of DNA fragments. Electroelution was used to move the DNA fragments from the agarose plug into a solution containing polyamines to protect the DNA from shear-induced breakage. The DNA was stained with the bisintercalating dye thiazole orange homodimer and introduced into our ultrasensitive flow cytometer. A histogram of the fluorescence intensities (fingerprint) was constructed. RESULTS: Gram-positive Bacillus globigii and gram-negative bacteria Escherichia coli and Erwinia herbicola were distinguished by the fingerprint pattern of restriction fragments of their genomic DNA. DNA sizes determined by FCM are in good agreement with pulsed-field gel electrophoresis (PFGE) analysis. Flow cytometry requires only picogram quantities of purified DNA and takes less than 10 min for data collection and analysis. When the total sample preparation time is included, the analysis times for PFGE and FCM are similar ( approximately 3 days). CONCLUSIONS: FCM is an attractive technique for the identification of bacterial species. It is more sensitive and potentially much faster than PFGE.     

The relevance of flow cytometry for biochemical analysis

Flow cytometry (FCM) allows the simultaneous measurement of multiple fluorescences and light scatter induced by illumination of single cells or microscopic particles in suspension, as they flow rapidly through a sensing area. In some systems, individual cells or particles may be sorted according to the properties exhibited. By using appropriate fluorescent markers, FCM is unique in that multiple structural and functional parameters can be quantified simultaneously on a single-particle basis, whereas up to thousands of biological particles per second may be examined. FCM is increasingly used for basic, clinical, biotechnological, and environmental studies of biochemical relevance. In this critical review, we summarize the main advantages and limitations of FCM for biochemical studies and discuss briefly the most relevant parameters and analytical strategies. Graphical examples of the biological information provided by multiparametric FCM are presented. Also, this review contains specific sections on flow cytoenzymology, FCM analysis of isolated subcellular organelles, and cell-free FCM.     

不同应激损伤所致血管内皮细胞中vWF的变化

目的：通过检测血管内皮细胞（ＶＥＣ）中ｖＷＦ的变化,评价ＶＥＣ的损伤。方法：利用免疫组化技术对体外培养的猪肺动脉内皮细胞（ＰＡＥＣ）和大鼠主动脉内皮细胞（ＡＥＣ）中ｖＷＦ进行免疫荧光标记,通过流式细胞仪对ｖＷＦ的细胞阳性率和荧光强度进行定性、定量分析,观察低氧和低温所引起的ｖＷＦ变化。结果：正常猪ＰＡＥＣ和大鼠ＡＥＣ的ｖＷＦ阳性率在８０％左右,阳性程序较高。但缺氧或冷冻损伤可使ＰＡＥＣ和ＡＥＣ的ｖ     

流式细胞仪在微型浮游植物生态学中的应用

微型浮游植物是水域生态系统中的初级生产者 ,是海洋生态系统存在的基础。习惯上浮游植物根据其粒径大小有分为小型浮游植物 (ｍｉｃｒｏ - ｐｈｙｔｏｐｌａｎｋｔｏｎ ,>2 0ｕｍ) ,微型浮游植物 (ｎａｎｏ - ｐｈｙｔｏｐｌａｎｋ ｔｏｎ ,<2 0ｕｍ ,>2ｕｍ )和超微型浮游植物( ｐｉｃｏ - ｐｈｙｔｏｐｌａｎｋｔｏｎ ,<2ｕｍ )三类[13 ] 。本文将后两类统称为微型浮游植物。在早期的多数研究中 ,检测技术和方法落后 :譬如测定水域中的生物量和叶绿素ａ含量常用的拖网方法 ,几乎丢失了所有的微型浮游植物 ;即使是实验室内常用 0 .45ｕｍ…     

Automated fluorescence image cytometry. DNA quantification and detection of chlamydial infections

Digitized fluorescence microscopy in conjunction with automated image segmentation is a promising approach for screening clinical specimens quickly and reliably. This paper describes the hardware and software of a prototype image-based cytometer that can identify fluorescent objects, discriminate true objects from artifacts and divide overlapping pairs of objects. The use of this image cytometer is discussed for: (1) the measurement of the DNA ploidy distribution of isolated mature rat liver nuclei labeled with 4',6-diamidine-2-phenylindole; (2) the comparison of the DNA ploidy distributions of the same samples measured by image cytometry (ICM) and flow cytometry (FCM); and (3) the quantification of chlamydial infection by double labeling cells with antichlamydiae antibody and Hoechst 33258 for nuclear DNA analysis. Ploidy distributions measured by the automated image cytometer compared favorably to those obtained by FCM. All pairs of overlapping nuclei were automatically detected by an additional computer algorithm, and those pairs that were clearly more than one nucleus by visual inspection were correctly divided. The irregular morphology of the chlamydiae-infected cells meant that 26% of them were not correctly identified in the fluorescein-stained images (as judged by manual inspection), but all cells were nevertheless detected correctly from the images of the Hoechst-stained samples. Automated fluorescence ICM yielded results similar to those obtained with FCM and had the additional benefit of maintaining cell and tissue architecture while preserving the opportunity for subsequent manual inspection of the specimen.     

Spectra of cells in flow cytometry using a vidicon detector.

A TV type vidicon detector was interfaced to a flow cytometer (FCM) to obtain spectra of fluorophores in cells during flow. The normal operations of the FCM are undisturbed. A spectrograph spreads 320 nm of the fluorophore fluorescence emission across the 500 channels of the detector. Spectra of fluorescamine (a surface labeling agent) and of propidium iodide (a nuclear stain) were obtained from Balb 3T3 cells, and the chlorophyll and phycobilin peaks were resolved from flowing blue-green algae in the FCM. Under typical flow conditions, operation of the vidicon in the continuous mode gives for these fluorophores a S/N of several hundred to one in approximately 3 sec. The vidicon was also gated to obtain spectra of single cells and of cells in selected portions of the cell cycle. For example, the spectrum of fluorescamine was obtained from cells in the G1 phase of the growth cycle by using as a gate trigger the FCM discriminator output derived from the propidium iodide signal.     

Increased growth yields of Mycoplasma spp. in the presence of pyruvate

Viable and non-viable African green monkey kidney (Vero) cells after treatment with Clostridium perfringens enterotoxin (CPE) followed by simultaneous double staining with fluorescein diacetate (FDA) and propidium iodide (PI) were counted with a flow cytometer (FCM). Within 1 min the FCM analysed 10 000 Vero cells in a sample for viability. After treatment of Vero cells with CPE for 60 min and staining with FDA-PI for 5 min, a reproducible dose-response curve was obtained between 25 and 400 ng/ml of CPE and percentage viable cell numbers. The FCM analysis proved to be a strong tool for rapid discrimination between viable and non-viable Vero cells treated with CPE in a large number of samples at a time.     

In vivo flow cytometry: a horizon of opportunities

Flow cytometry (FCM) has been a fundamental tool of biological discovery for many years. Invasive extraction of cells from a living organism, however, may lead to changes in cell properties and prevents studying cells in their native environment. These problems can be overcome by use of in vivo FCM, which provides detection and imaging of circulating normal and abnormal cells directly in blood or lymph flow. The goal of this review is to provide a brief history, features, and challenges of this new generation of FCM methods and instruments. Spectrum of possibilities of in vivo FCM in biological science (e.g., cell metabolism, immune function, or apoptosis) and medical fields (e.g., cancer, infection, and cardiovascular disorder) including integrated photoacoustic-photothermal theranostics of circulating abnormal cells are discussed with focus on recent advances of this new platform.