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细胞分选实验先容

编辑:   上传日期:2017-08-16  阅读次数:

一、细胞分选服务先容

细胞分选是据细胞的属性,将混合细胞分为具有不同特性的几个不同类群的方法。细胞分选常用的方法是流式细胞仪分选和免疫磁珠细胞分选。此技术广泛应用于免疫学、血液学、肿瘤学和神经生物学中。

二、实验原理

流式细胞分选是利用流式细胞分选仪,对细胞的物理、生理、生化、免疫、遗传、分子生物学性状及功能状态等进行定性或定量检测,它可根据发射光的荧光强度和波长将发光颗粒亚群分开并可实现单克隆分选,对复杂样本中的细胞进行鉴定、分类、定量和分离。免疫磁珠细胞分选是把细胞用超级顺磁性的 MACS MicroBeads (MACS微型磁珠)特异性地标记,磁性标记完后,把这些细胞通过一个放在强而稳定磁场中的分选柱。分选柱里的基质造成一个高梯度磁场。被磁性标记的细胞滞留在柱里而未被标记的细胞则流出。当分选柱移出磁场后,滞留柱内的磁性标记细胞就可以被洗脱出来,这样就完全可以获得标记和未标记的两个细胞组份。

三、实验流程

流式细胞分选
1. 取1×105个待检测的细胞,加5 ml DPBS洗涤,室温2000 r/min离心5 min,弃上清,然后用200 μl 0.5% BSA-DPBS重悬细胞;
2. 想细胞悬液中加入荧光染料标记的抗体,4℃孵育30 min;
3. 将染好的细胞用0.5% BSA-DPBS 洗涤两次,然后用500 μl 0.5% BSA-DPBS重悬,流式细胞仪检测;
4. 结果统计分析。
磁珠细胞分选方法
1. 离心收集待分离细胞,用少量PBE孵育液(0.5%BSA、0.08%EDTA,PH7.2),真空抽滤除菌及液体内气体,充分混悬细胞(0.5 ml/1×108细胞),加入一抗(10-20 μg/ml终浓度),4℃孵育30 min;
2. 用20倍体积PBE洗细胞一次,再加PBE(0.3 ml/1×108细胞)充分混悬细胞后,加入相应二抗包被的超微磁珠,混匀后置8~15℃孵育10~15 min;
3. 将分离柱安装入磁场中,加入0.5 ml PBE,在重力作用下自然流尽,以预处理分离柱。

细胞分选

Flow cytometry-Cell analysis(流式细胞分析)

Flow cytometry is a highly sensitive technology for single cell fluorescent signals measurements. By using specific fluorescent probes, this technology allows simultaneous multiparametric analysis of many thousands of cells per second, enabling researchers to rapidly analyze complex cell populations. 
Cells or particles labeled with fluorescent molecules enter a fluid stream and flow at a constant speed in flow cytometer. When the cells pass through laser focuses along the stream one by one, the fluorescent probes are excited by the laser of specific wavelength and then emit light. The optics collects and transduces fluorescent light to the detector according to its color. Then the fluorescent light is detected, amplified and translated into an electronic signal, which will be processed by signal electronics and sent to a computer for presentation. Information about the cell is recorded and the result can be visually displayed on the computer screen in real time.

Flow cytometry-Cell Sorting(流式细胞分选)

Cell sorting is the physical isolation and enrichment of selected cells. Sorting on a flow cytometer is executed just after the standard measuring process. During sorting process the stream is controllably vibrated at a specific frequency to stably break it into droplets. These droplets containing cells with selected values by investigator are then positively or negatively charged more or less and go through a constant electric field. As a result the charged droplets are sent along selected trajectories into vessels (tube or plate), and the uncharged fluid containing non-target cells flow into waste drawer.

When should you use flow cytometric cell sorting?(什么时候可以使用流式细胞分选?)

1). To enrich target cell population at a very high purity of 95%-100%.
2). To sort desired cells on the basis of multiple parameter measurements. 
3). To separate cell populations with some low expression antigens on cell surface.
4). To isolate cells according to the accurately quantitative density of specific cell markers. 
5). To acquire one single cell in multi-well plates.
6). To separate cells on the basis of internal constituents or functional staining, e.g. fluorescent hydrolysates. 
7). To detect and sort very rare cells (0.001% or less) from mixed populations.


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