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细胞内流式细胞术

Overview
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Intracellular flow cytometry is a powerful technique for the identification of cell types and the analysis of signaling and functional responses within cell lines and heterogeneous cell samples. For some cell types, such as Th17 and regulatory T cells (Tregs), definitive identification depends on the combined use of surface and intracellular markers such as cytokines or transcription factors. Intracellular flow cytometry also provides rich information concerning cellular function and signaling responses. Fluorescent antibodies specific for cell surface markers can be combined with markers of apoptosis, proliferation and protein phosphorylation to determine which cell subsets respond to various stimuli or treatments. The combined use of multiple markers decreases data acquisition time and conserves precious samples, since more parameters can be measured on a per-cell basis. While western blotting and other methods are useful for the examination of single proteins expressed by entire cell populations, flow cytometry allows the detection of multiple proteins simultaneously at the level of individual cells.

 

BD Biosciences provides fluorochrome-conjugated antibodies, buffers, kits and protocols to facilitate intracellular flow cytometry. Our antibodies are tested in biologically relevant model systems. These established tools enable new discoveries in fields such as immunology, inflammation and stem cell biology. Find the tools and techniques, including BD fluorochrome-conjugated antibodies, buffers, kits and protocols that support intracellular cytokine staining and phosphoprotein and transcription factor detection by intracellular flow cytometry.

 

Protocols for intracellular flow cytometry

 

 



细胞内流式细胞术是鉴定细胞类型和分析细胞系和异质性细胞样本内信号传导和功能反应的有力技术。对于一些细胞类型,如Th17和调节性T细胞(Treg),其最终鉴定依赖于细胞表面和细胞内标记物的组合使用,如细胞因子或转录因子。细胞内流式细胞术还可提供细胞功能和信号反应方面的丰富信息。细胞表面标记物的特异性荧光抗体可与凋亡、增殖和蛋白磷酸化标记物结合,以确定哪些细胞亚群对各种刺激或治疗有反应。多个标记物的联合使用可减少数据采集时间并保存宝贵样本,因为这样可以测定单个细胞的更多参数。虽然蛋白免疫印迹法和其他方法都可以检测整个细胞群表达的单个蛋白,但流式细胞术可在单个细胞水平同时检测多个蛋白。

 

BD生物科学公司提供荧光染料标记抗体、缓冲液、试剂盒和实验方案,以促进细胞内流式细胞术的实施。已在生物学相关模型系统测试了我们的抗体。这些已确定的工具能够让我们在免疫学、炎症和干细胞生物学等领域有新的发现。找到支持通过细胞内流式细胞术检测细胞内细胞因子染色以及磷蛋白质和转录因子的工具和技术,包括BD荧光染料标记抗体、缓冲液、试剂盒和实验方案。

 

 

细胞内染色的基本原理


虽然细胞表面染色技术是相对标准的,但是,细胞内标记物的最佳染色往往取决于目标蛋白的生物学特性。根据蛋白质在细胞内的位置、与其他分子的结合及其稳定性,推荐不同的细胞制备和染色方法。例如,细胞因子是典型的分泌蛋白。

 

但是,如果捕获在细胞内,可以使用蛋白转运抑制剂,如BD GolgiStop™(含莫能菌素)或BD GolgiPlug™(含布雷菲德菌素A),将它们染色为细胞内蛋白。使用BD Cytofix/ Cytoperm™固定和透化溶液进行温和固定和透化后可以相对容易地获得细胞因子。

 

相较于细胞因子,转录因子通常位于细胞核内,并与DNA和其他蛋白质结合。一些蛋白的磷酸化(如信号转导及转录激活因子5)会导致二聚体形成,这会掩盖相关的磷酸化表位。此外,细胞内的磷酸酶可以迅速使这些蛋白质去磷酸化。因此,处理后必须迅速固定细胞并进行更强的透化,以使抗体进入细胞核,并达到被破坏的分子复合物内的表位。

 

固定并透化细胞(用虚线膜表示),然后染色,并采用流式细胞术进行分析。为了研究细胞因子的分泌,首先用蛋白质运输抑制剂处理细胞,从而使靶蛋白在细胞内蓄积。

 

While techniques for cell surface staining are relatively standard, optimal staining for intracellular markers often depends on the biology of the target protein. Depending on the protein’s location inside the cell, association with other molecules, and its stability, different cell preparation and staining methods are recommended. Cytokines, for example, are typically secreted proteins.

 

However, if they are trapped inside the cell, they can be stained as intracellular proteins using protein transport inhibitors such as BD GolgiStop™ (containing monensin) or BD GolgiPlug™ (containing brefeldin A). Cytokines are relatively accessible using the gentle fixation and permeabilization afforded by BD Cytofix/ Cytoperm™ Fixation and Permeabilization Solution.

 

In contrast to cytokines, transcription factors often are localized inside the nucleus and bound to DNA and other proteins. Phosphorylation of some proteins, such as Stat5, results in dimer formation that masks the phosphorylated epitope of interest. Also, intracellular phosphatases can quickly dephosphorylate these proteins. Therefore, after treatment, cells must be quickly fixed and subjected to stronger permeabilization conditions to allow the antibody to enter the nucleus and access the epitope within disrupted molecular complexes.

 

Cells are fixed and permeabilized (symbolized by dashed line membrane), stained, and analyzed by flow cytometry. For studies on cytokine production, cells are first treated with a protein transport inhibitor to allow accumulation of the target protein inside the cell.

BD Biosciences provides tools to support intracellular flow cytometry

To facilitate intracellular flow cytometry assays, BD has developed several kits, buffers and protocols. In addition, many fluorescent antibodies specific for key cell surface markers have been tested in several buffer systems to save time, sample and money. Buffers are available for:

 

Detection of cytokines 

BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution (Cat. No. 554714) is suitable for staining most cytokines and cell surface markers. This buffer system can also be used to stain some transcription factors and other intracellular proteins. This buffer system contains mild detergents along with a formaldehyde-based fixative.

 

Transcription factors 

The BD Pharmingen™ Transcription Factor Buffer Set (Cat. No. 562574/562725) is designed for the staining of transcription factors alone or in combination with cell surface markers and cytokines. This buffer system contains mild detergents along with a formaldehyde-based fixative.

 

Detection of phosphorylated protein 

BD Phosflow™ Perm Buffer III (Cat. No. 558050) is the recommended permeabilization buffer for phosphoepitope detection by flow cytometry. Perm buffer III is a harsh alcohol-based buffer. Alternative permeabilization buffers also are available to accommodate particular experimental requirements. We also provide a convenient buffer compatibility tool, detailing many common clones and their compatibility with BD Phosflow™ Buffer protocols. 

 

The permeabilization technique used can negatively impact the detection of cell surface and other intracellular antigens. The same techniques that allow access to the nucleus and open up DNA/protein or protein/protein complexes can often denature cell surface antigens, preventing their detection by antibodies. While detection of different intracellular proteins might require different conditions, the basic principles are the same: cells are fixed and permeabilized and then stained intracellularly with fluorescent antibodies.

 

 

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STAT phosphotyrosine epitopes are obscured by dimerization within activated cells.

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Optimal cell permeabilization conditions vary by epitope location.

BD生物科学公司提供了实施细胞内流式细胞术的工具。

为了便于完成细胞内流式细胞术检测,BD公司还开发了几种试剂盒、缓冲液和实验方案。此外,已经在几个缓冲系统中对许多关键细胞表面标记物的特异性荧光抗体中进行测试,以节省时间、样品和资金。缓冲液可用于:

 

细胞因子的检测

BD Cytofix/Cytoperm™固定/透化液(目录号:554722)适用于染色大多数细胞因子和细胞表面标记物。该缓冲系统也可用于染色一些转录因子和其他细胞内蛋白。该缓冲系统含有温和的洗涤剂与甲醛基固定剂。

 

转录因子

BD Pharmingen™转录因子缓冲液(目录号:562574/562725)用于单独或联合细胞表面标记物和细胞因子对转录因子进行染色。该缓冲系统含有温和的洗涤剂与甲醛基固定剂。

 

磷酸化蛋白的检测

BD Phosflow™透化缓冲液III(目录号:558050)是推荐用于流式细胞术检测磷表位的透化缓冲液。透化缓冲液III是一种粗糙醇基缓冲液。对于特定的实验要求,也可使用其他符合要求的透化缓冲液。我们还提供了方便的缓冲液兼容工具,其中详细介绍了许多常见克隆及其与BD Phosflow™缓冲液实验方案的兼容性。

 

使用的透化技术会对细胞表面和其他细胞内抗原的检测产生负面影响。可以进入细胞核并打开DNA/蛋白质或蛋白质/蛋白质复合物的相同技术通常可以使细胞表面抗原变性,防止被抗体检测到。虽然检测不同的细胞内蛋白可能需要不同的条件,但基本原理是相同的,即固定并透化细胞,然后用荧光抗体在细胞内染色。

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活化细胞内的STAT磷酸络氨酸表位会被二聚作用掩盖。

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最佳胞透化条件因表位位置而异。

Cytokine Detection
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Transcription Factors
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Phosphorylation
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Multiplexing
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Resources
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仅供研究使用,不用于诊疗程序。

Alexa Fluor是Life Technologies Corporation公司的商标。CF是Biotium公司的商标。Cy是全球生命科学解决方案德国股份有限公司的商标,或者是以Cytiva名义开展业务的一家附属公司。Cytobank是美国贝克曼库尔特有限公司的商标。