多参数分析超抗原SEB诱导小鼠NKT细胞变化的流式细胞术

探讨采用四色荧光探针检测超抗原SEB诱导小鼠NKT细胞的多参数流式细胞术.选用培养C57BL/J小鼠脾淋巴细胞,四色免疫荧光标记FITC、PI、PerCP及APC,应用FACS Calibur型流式细胞仪,以Cellquest软件获取、分析及储存.经SEB诱导后流式细胞术可同时检测到淋巴细胞亚群及NKT细胞CD3 NK1.1 ,CD4 NK1.1 ,CD8 NK1.1 的变化.第10d时,CD3 /NK1.1 百分率达到最高.多参数的流式细胞术为研究NKT细胞功能提供一种快速、客观及全面多指标的检测手段.     

用荧光微球-流式细胞术检测流行性出血热的初步研究

发展了一种应用荧光微球进行流行性出血热流式细胞术检测的新方法.方法为先在荧光微球表面连接上流行性出血热特定的抗体以及相应的抗原等物质,然后,通过TEM(透射电子显微镜)和流式细胞仪等技术对实验结果进行了表征和解释.方法的进一步研究正在进行中.     

快速定量检测细菌的FCM系统研制及性能评估

为了实现对饮用水中细菌快速定量检测,建立了基于流式细胞术的高通量定量检测系统。对该系统的信号采集系统、绝对计数方法以及在细菌检测方面的综合性能进行了研究和评估。根据饮用水中典型细菌的荧光染料及其荧光激发光谱特点,介绍了流式细胞术快速检测细菌的工作原理及硬件平台。通过简化细菌的荧光信号强度计算模型,评估了信号采集系统的信噪比。建立了基于流量传感器的绝对计数方法,将检测系统与以参比微球法进行绝对计数的BD LSR Fortessa进行了一系列对比实验和统计学分析,测试和评估了检测系统在饮用水中细菌检测的综合性能水平。实验结果表明:对于4MHz宽带的荧光信号,信号采集系统的信噪比可达86dB;对于一定浓度内的微球,系统对它测试cv值低于2%,与BD仪器测试结果的相关系数高达0.999 6,对等比例稀释的微球测试线性度高达0.999 8,最低可检测细菌浓度可达10~2 particles/mL;系统对E.coli和S.aureus含量测试结果的cv值均低于7%,与BD仪器测试结果的相关系数均高于0.995 9,两仪器测试结果的相对误差均在4%以内。该仪器能实现对细菌的高精度快速定量检测,为饮用水中典型细菌快速检测仪器的开发提供了参考。     

荧光显微镜观察银杏内酯B对大鼠心肌细胞凋亡的影响

目的：建立一种简便、快速的细胞凋亡检测方法，探讨银杏内酯B（GB）对心肌细胞凋亡的影响。方法：体外培养大鼠心肌细胞，利用荧光显微镜观察经Hoeehst 33258染色的心肌细胞，记录凋亡细胞核形态学改变并计算凋亡百分率。结果：H2O2可诱导心肌细胞发生凋亡，与H2O2组相比，GB组大鼠心肌细胞凋亡率明显降低，并有显著性差异（P〈0．05）。结论：银杏内酯B对大鼠心肌细胞凋亡具有保护作用。     

检测早期细胞凋亡的流式细胞术

探讨了采用多参数流式细胞术分析氧化应激造成的心肌细胞凋亡过程中线粒体膜电位的改变。应用荧光探针JC-l可敏感检测到这种改变，是一种检测早期细胞凋亡改变的理想手段。     

富勒烯膦酸衍生物对HeLa细胞生长的影响

研究富勒烯膦酸衍生物2P对人宫颈癌HeLa细胞生长的影响.利用本实验室合成的富勒烯膦酸衍生物2P作用于HeLa细胞,并应用MTT法、流式细胞术、和PI/H033342双染色法等方法检测其效应.结果表明,2P在光照条件下对HeLa细胞生长具有明显的抑制作用,这种作用有剂量依赖性和光照依赖性.经过双染后荧光镜检测和流式检测后发现有高比例的凋亡细胞存在.流式细胞术结果还发现2P对HeLa细胞周期产生明显影响,表现为G1期细胞减少,S期和G2/M期细胞增多,推测细胞可能被2P阻断在S、G2期.以上结果提示富勒烯膦酸衍生物的细胞毒作用,以及作为抗肿瘤药物的可能.     

荧光编码微球—流式细胞和生化分析技术及其最新发展

简述了流式细胞术的发展历史和工作原理.对该技术与荧光编码微球技术相结合而发展起来的高通量细胞和生化分析技术及其最新发展作了评述.流式细胞术是一种对流动液体中排成单列的细胞逐个进行检测的技术,通过检测得到相应细胞的光散射和荧光信号,进而测定细胞的大小、形状、DNA、RNA、表面受体、酶活性、膜通透性和钙流量等,在癌症和爱滋病研究、新药开发、干细胞和基因治疗、遗传学、家畜性别选择、农作物新品种开发等方面都有广泛应用.采用顺序进样技术,则可不必加压而实现少量样品的自动分析和作亚秒水平分子相互作用的动力学测量.将该技术与近年发展起来的荧光染料编码微球技术相结合,则成为一种可以与生物芯片技术相媲美的多组分同时分析技术.采用微流控芯片技术和发光半导体量子点编码微球技术,进一步改善了方法的适用性,某些主要性能甚至超过了生物芯片技术,有望从研究实验室逐步进入临床实验室,最终进入病房和寻常百姓家,成为防病、诊断和监护病人的重要手段.     

PerkinElmer推出激光诱导活细胞动态过程快速示踪装置

PerkinElmer公司最近推出一种激光诱导活细胞动态过程快速示踪装置。该装置可附加在著名的UltraView ERS或RS系列活细胞高速成像系统上。采用该装置，可完成活细胞样本上多达100个目标区域（ROI，region of interest）的光漂白后荧光恢复（FRAP）fluorescence recovery after photobleaching）、在光漂白过程中的荧光损失（FLIP，fluorescence loss in photobleaching）、光诱导开关（Photoswitching，即用特定波长的激光激发特定的荧光蛋白。使得该荧光蛋白的发射波长发生变化），和光诱导激活（Photoactivation，即通过激光诱导，点亮特定的荧光蛋白）的观测示踪工作。通过灵活的运用这些技术和方法，     

蛋白质／多肽的毛细管电泳-激光诱导荧光检测分析方法发展

毛细管电泳（CE）与激光诱导荧光检测器（LIF）联用为蛋白质／多肽类样品提供了一种快速、灵敏的分析方法。选择并开发合适的荧光衍生化试剂,建立并优化相应的衍生、分离过程,使用并发展高性能、低成本的激光诱导荧光检测器件是实现高效分离、高灵敏度检测的有效途径。本文将各荧光衍生化试剂以激发光源归类,在探讨常用荧光衍生化试剂特性的基础上,对近几年蛋白质／多肽类样品的毛细管电泳-激光诱导荧光分析方法发展加以系统综述。     

基于液芯波导激光诱导荧光检测器的缝管自动进样毛细管电泳仪的研制及其在DNA分析中的应用

介绍一种自行研制的毛细管电泳仪。以半导体激光器作为激发光源,使用液芯波导石英毛细管同时作为电泳分离通道和荧光检测光路,结合基于取样探针、缺口型样品圆盘两者的缝管自动进样系统,以激光诱导荧光的检测方式实现对两种常用DNA分子标记物（DNA Marker）快速、低耗、准确的有效检测。该仪器具有结构简单、体积小、操作方便等特点。     

Rapid assay for pathogenic salmonella organisms by immunofluorescence flow cytometry

Multi-parameter flow cytometry was investigated for the rapid detection of specific serotypes of salmonellas (S. typhimurium and S. montevideo) labelled with fluorescent monoclonal antibodies, both in pure culture and in a typical food matrix (full-fat milk). In all cases, the method was accurate to levels of below 10⁴ target cells per ml for a total assay time of about 30 min. After 6 h non-selective enrichment in the presence of a 10 000-fold excess of competing micro-organisms (Escherichia coli) the corresponding detection limit was about 20 cells ml^?1. These results suggest that flow cytometry has significant potential for the detection of pathogenic micro-organisms in the food industry.     

Automated classification and visualization of fluorescent live cell microscopy images

Robotic, high‐throughput microscopy is a powerful tool for small molecule screening and classifying cell phenotype, proteomic and genomic data. An important hurdle in the field is the automated classification and visualization of results collected from a data set of tens of thousands of images. We present a method that approaches these problems from the perspective of flow cytometry with supporting open‐source code. Image analysis software was created that allowed high‐throughput microscopy data to be analysed in a similar manner as flow cytometry. Each cell on an image is considered an object and a series of gates similar to flow cytometry is used to classify and quantify the properties of cells including size and level of fluorescent intensity. This method is released with open‐source software and code that demonstrates the method's implementation. Accuracy of the software was determined by measuring the levels of apoptosis in a primary murine myoblast cell line after exposure to staurosporine and comparing these results to flow cytometry.     

流式细胞术快速检测T细胞亚群和NKT细胞亚群的免疫荧光变化

利用单克隆抗体与免疫荧光结合的特异性,探讨流式细胞仪检测T细胞亚群和NKT细胞亚群免疫荧光变化。选用培养C57BL/7小鼠脾细胞分别经SEB.COA活化,收集体外扩增10d淋巴细胞为效应细胞,经免疫荧光探针FITC、PI、PerCP和APC标记可同时检测各亚群免疫荧光变化,结果表明SEB活化主要是NKT细胞,它们与COA活化NKT细胞亚群相比比率显著增高。COA活化细胞主要是T细胞亚群。依据免疫荧光原理应用流式细胞术,为进一步研究NKT细胞结构和功能提供一种快速的检测手段。     

Image cytometric method for quantifying the relative amount of DNA in bacterial nucleoids using Escherichia coli

An image cytometric method for quantifying integrated fluorescence was developed to measure the relative DNA contents of bacterial nucleoids. Image analysis was performed with newly developed macros in combination with the program Object-Image, all downloadable from http://simon.bio.uva.nl/object-image.html. Four aspects of the method were investigated. (i) Good linearity was found over a ten-fold range of fluorescence intensity in a test with a calibration kit of fluorescent latex spheres. (ii) The accuracy of the method was tested with a narrowly distributed Escherichia coli population, which was obtained by growing cells into stationary phase. The width of the image cytometric distribution was approximately 6%, in good agreement with results obtained by flow cytometry. (iii) The error contribution of manual focusing could be kept below 2%, although a strong dependency between integrated fluorescence and focus position was observed. (iv) The results were verified with a flow cytometer, which gave similar distributions for the DNA contents per cell expressed in chromosome equivalents (4.8 fg of DNA). We used the presented method to evaluate whether the DNA conformation had any effect on the total fluorescence of bacterial nucleoids. Experiments using nucleoids with the same amount of DNA in either a dispersed or a compact conformation showed no significant difference in integrated fluorescence, indicating that it is possible to determine the DNA content per nucleoid independently of the actual organization of the DNA.     

低能量激光照射对受张力的成骨细胞周期和胞内Ca~(2+)浓度的影响

对成骨样细胞施加了周期性张力,探讨了成骨细胞MG-63受张力时的细胞周期和胞内Ca2+浓度的变化规律以及低能量激光照射(LLLI)对此变化规律的影响,揭示了LLLI促进骨形成的机制。实验首先将MG-63细胞随机分成对照组、加力组、激光加力组3组,用流式细胞术检测各组细胞周期。然后,将MG-63细胞分为张力组和激光张力组两组,对胞内Ca2+浓度变化进行测定,对2组细胞分别加力0,5,15,30,60min,再对激光张力组施加激光照射1min后收取细胞,并用荧光探针Fluo-3-AM测定成骨样细胞内Ca2+浓度和Ca2+阳性细胞百分比。结果显示:MG-63细胞加载张力后,细胞增殖指数提高,施加LLLI后受力的成骨细胞增殖指数进一步提高。张力引起胞内Ca2+浓度增加,变化剧烈,LLLI使变化曲线平缓,且胞内Ca2+浓度和Ca2+阳性细胞百分比增加。实验结果表明:LLLI可促进受张力的成骨细胞增殖,推测可能是通过调节成骨细胞内Ca2+浓度的变化规律和水平来完成的。     

Analysis of isolation of cerebral cortical neurons in rats by different methods

The aim of this study was to find a way to efficiently separate neuronal cells from the cerebral cortex of adult rats, providing a reference method for rapid acquisition of neuronal cells from the adult rat brain. Fifteen SD rats were randomly divided into three groups, with five SD rats in each group. Then, neuron cells were isolated from the adult rat cerebral cortex by the grinding method, the trypsin method, and the collagenase II method, respectively. The expression of anti-NeuN in the neurons of each group was analyzed by flow cytometry. The acquisition rates and morphology of neurons of each group were observed by immunofluorescence staining. The grinding or collagenase II method is more suitable for rapid acquisition of neuronal cells from an adult rat’s cerebral cortex. The number of neuron cells obtained by the trypsin method were very few, so it is not convenient for later experiments.     

ICP-MS同时测定玛咖中16种稀土元素的研究

建立一种快速消解并同时测定玛咖中稀土元素的方法。采用微波消解样品,选择In作内标,电感耦合等离子体质谱法(ICP-MS)测定玛咖中稀土元素。对于所测的元素,校准曲线的相关系数>0.9990,回收率范围91.0%~111.0%,相对标准偏差0.4%~3.7%。该方法简便、快速、准确,灵敏度高,可用于玛咖中稀土元素的测定。     

In vivo remineralization of acid-etched enamel in non-brushing areas as influenced by fluoridated orthodontic adhesive and toothpaste

This study aimed to evaluate the in vivo remineralization of acid-etched enamel in non-brushing areas as influenced by fluoridated orthodontic adhesive and toothpaste. One hundred and twenty teeth from 30 volunteers were selected. The teeth were assigned to four treatments: no treatment (negative control); 37% phosphoric acid-etching (PAE) (positive control); PAE + resin-modified glass ionomer cement (RMGIC); and, PAE + composite resin. Patients brushed teeth with fluoridated (n = 15) or non-fluoridated (n = 15) toothpastes, so that etched enamel was protected with screens and it was not in contact with the brush bristles. Remineralization was evaluated by means of laser fluorescence (LF), environmental scanning electronic microscopy, and energy dispersive spectrometry after extraction. The LF means were compared by means of Wilcoxon and Mann Whitney tests. Environmental scanning electron microscopy scores were compared among the groups using a Kruskal Wallis test, whereas the Ca/P ratio was evaluated by means of an Analysis of Variance with subparcels (treatments) and Tukey's post-hoc test. There were no statistically significant differences between the tooth pastes and between the orthodontic adhesives evaluated. Most teeth presented only partial enamel remineralization. Therefore, the fluoride released by the RMGIC was not enough to cause increased crystal regrowth in the acid-etched enamel. The use of fluoridated toothpaste did not provide positive additional effect.     

应用流式细胞术对啤酒酵母在含油酸培养基中生长的定量研究

本研究采用流式细胞术测定啤酒酵母中的绿色荧光蛋白的表达量和细胞的存活度,提出一种可以用于监测啤酒酵母生长的快捷方法,筛选出较佳细胞培养方案,测定酵母细胞在不同含油酸培养基中的死亡速度常数。