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細(xì)胞分選實(shí)驗(yàn)介紹

2017-08-16
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一、細(xì)胞分選服務(wù)介紹

細(xì)胞分選是據(jù)細(xì)胞的屬性,將混合細(xì)胞分為具有不同特性的幾個(gè)不同類群的方法。細(xì)胞分選常用的方法是流式細(xì)胞儀分選和免疫磁珠細(xì)胞分選。此技術(shù)廣泛應(yīng)用于免疫學(xué)、血液學(xué)、腫瘤學(xué)和神經(jīng)生物學(xué)中。

二、實(shí)驗(yàn)原理

流式細(xì)胞分選是利用流式細(xì)胞分選儀,對(duì)細(xì)胞的物理、生理、生化、免疫、遺傳、分子生物學(xué)性狀及功能狀態(tài)等進(jìn)行定性或定量檢測(cè),它可根據(jù)發(fā)射光的熒光強(qiáng)度和波長(zhǎng)將發(fā)光顆粒亞群分開并可實(shí)現(xiàn)單克隆分選,對(duì)復(fù)雜樣本中的細(xì)胞進(jìn)行鑒定、分類、定量和分離。免疫磁珠細(xì)胞分選是把細(xì)胞用超級(jí)順磁性的 MACS MicroBeads (MACS微型磁珠)特異性地標(biāo)記,磁性標(biāo)記完后,把這些細(xì)胞通過一個(gè)放在強(qiáng)而穩(wěn)定磁場(chǎng)中的分選柱。分選柱里的基質(zhì)造成一個(gè)高梯度磁場(chǎng)。被磁性標(biāo)記的細(xì)胞滯留在柱里而未被標(biāo)記的細(xì)胞則流出。當(dāng)分選柱移出磁場(chǎng)后,滯留柱內(nèi)的磁性標(biāo)記細(xì)胞就可以被洗脫出來,這樣就完全可以獲得標(biāo)記和未標(biāo)記的兩個(gè)細(xì)胞組份。

三、實(shí)驗(yàn)流程

流式細(xì)胞分選
1. 取1×105個(gè)待檢測(cè)的細(xì)胞,加5 ml DPBS洗滌,室溫2000 r/min離心5 min,棄上清,然后用200 μl 0.5% BSA-DPBS重懸細(xì)胞;
2. 想細(xì)胞懸液中加入熒光染料標(biāo)記的抗體,4℃孵育30 min;
3. 將染好的細(xì)胞用0.5% BSA-DPBS 洗滌兩次,然后用500 μl 0.5% BSA-DPBS重懸,流式細(xì)胞儀檢測(cè);
4. 結(jié)果統(tǒng)計(jì)分析。
磁珠細(xì)胞分選方法
1. 離心收集待分離細(xì)胞,用少量PBE孵育液(0.5%BSA、0.08%EDTA,PH7.2),真空抽濾除菌及液體內(nèi)氣體,充分混懸細(xì)胞(0.5 ml/1×108細(xì)胞),加入一抗(10-20 μg/ml終濃度),4℃孵育30 min;
2. 用20倍體積PBE洗細(xì)胞一次,再加PBE(0.3 ml/1×108細(xì)胞)充分混懸細(xì)胞后,加入相應(yīng)二抗包被的超微磁珠,混勻后置8~15℃孵育10~15 min;
3. 將分離柱安裝入磁場(chǎng)中,加入0.5 ml PBE,在重力作用下自然流盡,以預(yù)處理分離柱。

細(xì)胞分選

Flow cytometry-Cell analysis(流式細(xì)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(流式細(xì)胞分選)

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?(什么時(shí)候可以使用流式細(xì)胞分選?)

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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