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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











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。






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.




STAT phosphotyrosine epitopes are obscured by dimerization within activated cells.


Optimal cell permeabilization conditions vary by epitope location.





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。



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。



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。







Cytokine Detection
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ELISA和BD®Cytometric Bead Array(CBA)等方法可测定全细胞群产生的分泌蛋白。相比而言,细胞内流式细胞术可分析相关细胞群内单一表型鉴定细胞类型分泌的细胞因子和其他炎症介质。




由于细胞因子通常是分泌蛋白质,必须首先使用蛋白质运输抑制剂将其捕获在细胞内。两种最常用的蛋白转运抑制剂为莫能菌素(BD GolgiStop™抑制剂)和布雷菲德菌素A(BD GolgiPlug™抑制剂)。莫能菌素可与高尔基体跨膜Na++/H+转运相互作用阻止蛋白质的分泌,而布雷菲德菌素A将细胞内分泌的蛋白质从cis/高尔基体中间复合物重新分配到内质网。2因此,蛋白质转运抑制剂的最佳选择因细胞因子和物种而异(表1)。




在存在布雷菲德菌素A的情况下,用葡萄球菌肠毒素B刺激外周血单核细胞6小时。刺激后,使用BD Cytofix/Cytoperm™缓冲系统固定和透化细胞。使用以下荧光抗体染色细胞:CD3 FITC、CD4 PerCP-Cy5.5、CD8 BD Horizon Brilliant Violet™ 421、IFN-γ PE和IL-2 APC。然后使用BD FACSVerse™流式细胞仪对细胞进行清洗和分析。

Species Cytokines Transport Inhibitor
Human IL-1α, IL-6, IL-8, TNF-α Monensin
Human IFN-γ, IL-2, IL-10, IL-12, MCP-1, MCP-3, MIG, MIP-1α, RANTES Either Monensin or Brefeldin A
Mouse IL-6, IL-12, TNF-α Brefeldin A
Mouse GM-CSF, IL-3, IL-4, IL-5, IL-10 Monensin
Mouse IFN-γ, IL-2 Either Monensin or Brefeldin A

IFN-γ and IL-2 production in CD8+ cells

PBMCs were stimulated with staphylococcal enterotoxin B for 6 hours in the presence of brefeldin A. After stimulation, cells were fixed and permeabilized using the BD Cytofix/Cytoperm™ Buffer System. Cells were stained with the following fluorescent antibodies: CD3 FITC, CD4 PerCP-Cy5.5, CD8 BD Horizon Brilliant Violet™ 421, IFN-γ PE and IL-2 APC. Cells were then washed and analyzed using the BD FACSVerse™ Flow Cytometer.


BD Biosciences has simplified the detection of cytokines and cell surface markers with kits, buffer systems and rich panels of fluorescent antibodies. Antibodies to surface markers and cytokines conjugated to a variety of fluorochromes are available. This allows for flexibility in staining panel design, supporting high content multicolor flow cytometric analyses to gain the most data from precious cell samples.


BD FastImmune™ Kits contain tested cocktails of fluorescent antibodies, an appropriate protein transport inhibitor, compatible buffers and a detailed protocol for the optimal preparation, staining and detection of cytokine-producing cells from whole blood. Onboard assays on the BD FACSVerse™ Flow Cytometer System further simplify the process.


BD Cytofix/Cytoperm™ Buffer has been cited in thousands of publications for the analysis of cytokine-producing cells by flow cytometry. Researchers select the protein transport inhibitor best suited to their cytokine of interest and use the BD Cytofix/Cytoperm™ Buffer System to fix, permeabilize and stain their cells for flow cytometric analysis.


Antigen-specific IFN-γ production by cytomegalovirus (CMV) pp65-stimulated CD4+CD69+ T lymphocytes.  


  1. Seder RA, Darrah PA, Roederer M. T-cell quality in memory and protection: implications for vaccine design. Nat Rev Immunol. 2008;8(4):247-258. doi: 10.1038/nri2274

  2. Schuerwegh AJ, Stevens WJ, Bridts CH, De Clerck LS. Evaluation of monensin and brefeldin A for flow cytometric determination of interleukin-1 beta, interleukin-6, and tumor necrosis factoralpha in monocytes. Cytometry. 2001;46(3):172-176. doi: 10.1002/cyto.1102




BD FastImmune™试剂盒包含经过测试的荧光抗体混合剂、合适的蛋白运输抑制剂、兼容的缓冲液和详细的实验方案,以完成全血细胞因子分泌细胞的最优制备、染色和检测。BD FACSVerse™流式细胞仪系统上的板载分析进一步简化了该过程。


BD Cytofix/Cytoperm™Buffer已被数以千计的出版物引用,用于流式细胞术分析细胞因子分泌细胞。研究人员选择最适合他们所关注细胞因子的蛋白转运抑制剂,并使用BD Cytofix/Cytoperm™缓冲系统来固定、透化和染色他们的细胞,用于流式细胞术分析。


巨细胞病毒(CMV)pp65刺激的CD4+CD69+ T淋巴细胞分泌的抗原特异性IFN-γ

Transcription Factors
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FoxP3被认为是调节性T细胞的主要调控因子。和许多转录因子一样,FoxP3可与数千个基因结合,进而上调或下调调节性T细胞的所需基因表达。3 SATB1(一个基因组组织者)会被FoxP3抑制,阻止T效应细胞因子(包括IL-4和IFN-γ)的诱导4






细胞的固定和透化会影响细胞表面标记物染色,这就使得选择兼容的缓冲液更为关键。为了进一步实现转录因子检测,BD生物科学公司还开发了BD Pharmingen™转录因子缓冲液。该缓冲液能充分透化细胞,暴露核内表位,同时还与许多细胞类型、细胞表面染色和串联染料兼容。




当多能干细胞分化为不同的细胞类型时,转录因子和其他蛋白的表达会发生变化。在哺乳动物胚胎发育过程中,定形内胚层会形成肝脏、胰腺和肠道。5,6 在进入最终内胚层的过程中,转录因子Sox17和FoxA2以及细胞表面标记物CD184(CXCR4)的水平会升高,而多能性标记物Nanog和Sox2的水平则下降8




为了便于研究干细胞中的转录因子,BD公司开发了几种检测关键干细胞特异性转录因子的试剂盒,包括BD Stemflow™人多能干细胞转录因子分析试剂盒(目录号:560589)、BD Stemflow™小鼠多能干细胞转录因子分析试剂盒(目录号: 560585)、BD Stemflow™人神经细胞谱系分析试剂盒(目录号:561526)以及BD Stemflow™人定形和胰腺内胚层分析试剂盒(目录号:562496)。这些试剂盒包含表征多能干细胞并跟踪多能干细胞分化至各自谱系的抗体和缓冲系统。



To facilitate the study of transcription factors in stem cells, BD has developed several kits for the detection of key stem cell–specific transcription factors including the BD Stemflow™ Human Pluripotent Stem Cell Transcription Factor Analysis Kit (Cat. No. 560589), the BD Stemflow™ Mouse Pluripotent Stem Cell Transcription Factor Analysis Kit (Cat. No. 560585), the BD Stemflow™ Human Neural Cell Lineage Analysis Kit (Cat. No. 561526), and the BD Stemflow™ Human Definitive and Pancreatic Endoderm Analysis Kit (Cat. No. 562496). These kits contain antibodies and buffer systems to characterize pluripotent stem cells and track the differentiation of pluripotent stem cells into respective lineages.


  1. Birzele F, Fauti T, Stahl H, et al. Next-generation insights into regulatory T cells: expression profiling and FoxP3 occupancy in Human. Nucleic Acids Res. 2011;39(18):7946-7960. doi: 10.1093/nar/gkr444

  2. Beyer M, Thabet Y, Müller RU, et al. Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation. Nat Immunol. 2011;12(9):898-907. doi: 10.1038/ni.2084

  3. Wang P, McKnight KD, Wong DJ, et al. A molecular signature for purified definitive endoderm guides differentiation and isolation of endoderm from mouse and human embryonic stem cells. Stem Cells Dev. 2012;21(12):2273-2287. doi: 10.1089/scd.2011.0416

  4. Takayama K, Inamura M, Kawabata K, et al. Efficient and directive generation of two distinct endoderm lineages from human ESCs and iPSCs by differentiation stage-specific SOX17 transduction. PLoS One. 2011;6(7):e21780. doi: 10.1371/journal.pone.0021780

  5. Murry CE, Keller G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell. 2008;132(4):661-680. doi: 10.1016/j.cell.2008.02.008

  6. D’Amour KA, Agulnick AD, Eliazer S, Kelly OG, Kroon E, Baetge EE. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol. 2005;23(12):1534-1541. doi: 10.1038/nbt1163
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BD Phosflow™产品包括一个优化的缓冲液和荧光单克隆抗体系统,用于流式细胞术检测细胞内信号分子和特定翻译后修饰。为了使用流式细胞术检测特异性磷酸化表位,需固定细胞来维持信号蛋白的磷酸化状态,然后将细胞透化使抗体进入细胞并与靶蛋白特异性结合。然后,对染色细胞的流式细胞术分析可捕获细胞内信号传导和蛋白磷酸化的快照。



Multiparameter flow cytometry offers key advantages for the detection and analysis of intracellular signaling within cells and cell subsets in complex mixtures. Since cell signaling studies often examine cellular responses to treatment with stimulatory or inhibitory molecules, unstimulated or untreated cells often provide the best controls for evaluating background staining. Unlike an immunoglobulin isotype control, an unstimulated cell control accounts for the unique background characteristics of each antibody as well as the basal phosphoprotein expression levels within the cells of interest. Cellular permeabilization is required to expose phosphorylated epitopes. Although multiple buffer systems are available, BD Phosflow™ Perm Buffer III is recommended for most applications. The BD Biosciences website contains useful information about the performance of various BD cell surface marker and intracellular antibodies in different buffer conditions and staining protocols.


BD Phosflow™ Kits provide useful tools for analyzing the signaling responses elicited by cells of the adaptive and innate immune systems. These kits include fluorescent antibody cocktails specific for cell surface markers to identify specific leucocyte subsets as well as fluorescent antibodies to key phosphoproteins. In addition, the kits provide compatible buffers, positive and negative control cells, and detailed protocols. The BD Phosflow™ Human Monocyte/NK Cell Activation Kit (Cat. No. 562089) allows the simultaneous study of protein phosphorylation in B cells, T cells, monocytes and NK cells from human whole blood, while the BD Phosflow™ Human T-Cell Activation Kit (Cat. No. 560750) is useful for the study of phosphoprotein responses in CD4 versus CD8 T cells.


多参数流式细胞术在检测和分析复杂混合物中的细胞和细胞亚群的细胞内信号传导时具有关键优势。由于细胞信号研究经常检查细胞对刺激性或抑制性分子处理的反应,未刺激或未处理的细胞通常是评价背景染色的最佳对照。与免疫球蛋白同型对照不同,未刺激细胞对照可以解释每种抗体的独特背景特征以及相关细胞内的基础磷蛋白质表达水平。需要通过细胞透化来暴露磷酸化的表位。虽然有多种缓冲系统可以使用,但大多数应用场景都推荐使用BD Phosflow™透化缓冲液III。BD生物科学公司网站包含不同缓冲条件和染色方案下各种BD细胞表面标记物和细胞内抗体的性能方面的有用信息。


Phosflow™试剂盒是分析适应性和先天免疫系统细胞所引发的信号反应的有用工具。这些试剂盒包括识别特定白细胞亚群的细胞表面标记物的特异性荧光抗体混合剂,以及关键磷蛋白质的特异性荧光抗体。此外,该试剂盒还提供兼容的缓冲液、阳性和阴性对照细胞以及详细的实验方案。BD Phosflow™人单核细胞/NK细胞激活试剂盒(目录号:562089)可同时研究人全血中B细胞、T细胞、单核细胞和NK细胞的蛋白磷酸化,而Phosflow™人T细胞激活试剂盒(目录号:560750可用于研究CD4和CD8+T细胞的磷蛋白质反应。

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在本例中,在固定、透化并使用表面和细胞内标记物染色之前,先用浓度递增的人重组IL-2刺激低温保存的外周血单核细胞。使用BD Pharmingen™转录因子Phospho Buffer Set可以同时检测对IL-2响应的调节性T细胞中的磷酸化信号转导及转录激活因子5和转录因子FoxP3。





在所示实验结果中,细胞分离自BALB/c小鼠胸腺和脾脏。胸腺细胞或脾细胞表面用CD44、CD62L、CD196或适当免疫球蛋白同型对照品的特异性荧光抗体染色。首先用BD Pharmingen™转录因子缓冲液固定和透化细胞,然后用RORγt、Foxp3、IL-17A和IFN-γ特异性荧光抗体进行细胞内染色。


在上图中比较了IL-17A的细胞表达与RORγt、FoxP3(调节性T细胞转录因子)、IFN-γ(Th1细胞因子)的表达。正如预期的那样,胸腺细胞中IL-17A+ RORγt+双阳性细胞较多,而IL-17A与Foxp3或IFN-γ基本没有共表达。脾细胞仅表达了很少的IL-17A。






  1. Ghoreschi K, Laurence A, Yang XP, Hirahara K, O’Shea JJ. T helper 17 cell heterogeneity and pathogenicity in autoimmune disease. Trends Immunol. 2011;32(9):395-401. doi: 10.1016/

  2. Ivanov II, McKenzie BS, Zhou L, et al. The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell. 2006;126(6):1121-1133. doi: 10.1016/j.cell.2006.07.035
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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。