| Immunophenotyping and Intracellular Staining of Fixed Whole Blood for Mass Cytometry (CyTOF)
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Author:
Date:
2020-09-05
[Abstract] In this report, we present the implementation of mass cytometry for intracellular staining using fixed whole blood. In our assay described here, 250 µl of whole blood, is stimulated in vitro with PMA/ionomycin (or left unstimulated), in the presence of secretion inhibitors (brefeldin A and monensin), lysed-fixed using SMART TUBE buffers, barcoded (optional), surface stained, fixed, stained for intracellular markers, fixed and DNA stained. Using 250 µl of whole blood from a healthy donor, we show that the expression of major lineage populations such as T cells, B cells, NK cells and monocytes, as well as cytokines such as CD4+ and CD8+ IFNγ and TNFα across multiple batches (n = 27) is consistent, with the co-efficient of variation (CVs) ≤ 21%, implying ...
[摘要] [摘要] 在本报告中，我们介绍了使用固定全血在细胞内染色中进行细胞计数的方法。在此处所述的测定中，在存在分泌抑制剂（布雷菲德菌素A和莫能菌素）的情况下，用PMA /离子霉素体外刺激（或不刺激）250 µl全血，使用SMART TUBE缓冲液裂解固定，加条形码（可选） ），表面染色，固定，细胞内标记物染色，固定和DNA染色。使用250微升的全血从健康供体中，我们表明，主要的谱系种群，例如T细胞，B细胞，NK细胞和单核细胞，以及细胞因子，例如CD4的表达+ 和CD8 + IFN γ 和TNF α 多个批次（n = 27）之间的一致性是一致的，变异系数（CV）≤21 ％，这意味着最小的变异性。对于每种主要细胞类型，该百分比报告为单峰百分比。据报道，响应刺激的细胞因子表达的百分比为直接亲代细胞类型的百分比。该方案具有许多好处：从生物学的角度来看，它可以应用于临床研究，尤其是在抽血量有限的情况下。从技术上讲，该协议可适用于条形码，这增加了更均匀的样品染色以及抗体保守性的好处，特别是对于大型研究人群。最后，对于包括当前全球COVID-19大流行在内的涉及传染病的研究，该方案允许在处理和染色之前固定传染性样品，从而降低了生物安全风险。
[背景 ] ...
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| Intracellular Cytokine Staining (ICS) on Human Lymphocytes or Peripheral Blood Mononuclear Cells (PBMCs)
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Author:
Date:
2015-04-05
[Abstract] Production of cytokines plays an important role in the immune response. Cytokines are involved in many different pathways including the induction of many anti-viral proteins by IFN gamma, the induction of T cell proliferation by IL-2 and the inhibition of viral gene expression and replication by TNF alpha. Cytokines are not preformed factors but are rapidly produced and secreted in response to cellular activation. Intracellular cytokine detection by flow cytometry has emerged as the premier technique for studying cytokine production at the single-cell level. It detects the production and accumulation of cytokines within the endoplasmic reticulum after cell stimulation, allowing direct TH1 versus TH2 determination. It can also be used in combination with other flow cytometry protocols for ...
[摘要] 细胞因子的产生在免疫应答中起重要作用。细胞因子参与许多不同的途径，包括通过IFNγ诱导许多抗病毒蛋白，通过IL-2诱导T细胞增殖以及抑制病毒基因表达和TNFα的复制。细胞因子不是预先形成的因子，但是响应于细胞激活而快速产生和分泌。通过流式细胞术的细胞内细胞因子检测已经成为研究单细胞水平的细胞因子产生的首要技术。它检测细胞刺激后细胞因子在内质网内的产生和积累，允许直接TH1对TH2测定。它也可以与其他流式细胞术协议结合使用细胞表面标记或与MHC多聚体进行免疫分型，以检测抗原特异性反应，使其成为一种非常灵活和通用的方法。这种能力，结合仪器的高通量性质，使细胞内细胞因子染色与现有的单细胞技术如ELISPOT，有限稀释和T细胞克隆相比具有巨大的优势。细胞内细胞因子染色的主要步骤如下：
1。使用特异性肽或非特异性活化混合物将细胞活化几小时;
2。加入蛋白质转运抑制剂（例如布雷菲德菌素A）以将细胞因子保留在细胞内;
。接下来，加入EDTA以从活化容器中除去粘附细胞;
4。洗涤后，可将细胞表面标记的抗体加入细胞中;
5。然后将细胞固定在多聚甲醛中并透化;
6。加入抗细胞因子抗体，并且可以通过流式细胞仪分析细胞。
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| Quantification of T Cell Antigen-specific Memory Responses in Rhesus Macaques, Using Cytokine Flow Cytometry (CFC, also Known as ICS and ICCS): from Assay Set-up to Data Acquisition
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Author:
Date:
2014-04-20
[Abstract] What was initially termed ‘CFC’ (Cytokine Flow Cytometry’) is now more commonly known as ‘ICS’ (Intra Cellular Staining), or less commonly as ‘ICCS’ (Intra Cellular Cytokine Staining). The key innovations were use of an effective permeant (allowing intracellular staining), and a reagent to disrupt secretion (trapping cytokines, thereby enabling accumulation of detectable intracellular signal). Because not all researchers who use the technique are interested in cytokines, the ‘ICS’ term has gained favor, though ‘CFC’ will be used here.
CFC is a test of cell function, exposing lymphocytes to antigen in culture, then measuring any cytokine responses elicited. Test cultures are processed so as to stain cells with monoclonal antibodies tagged with fluorescent markers, and to ...
[摘要] 最初称为"CFC"（细胞因子流式细胞术）的现在更通常称为"ICS"（细胞内染色），或更不常称为"ICCS"（细胞内细胞因子染色）。关键的创新是使用有效的渗透物（允许细胞内染色）和试剂破坏分泌（捕获细胞因子，从而能够累积可检测的细胞内信号）。因为不是所有使用该技术的研究人员都对细胞因子感兴趣，'ICS'术语已经受到青睐，尽管这里将使用"CFC"。
CFC是细胞功能的测试，将淋巴细胞暴露于培养物中的抗原，然后测量任何诱导的细胞因子应答。处理测试培养物以用用荧光标记物标记的单克隆抗体染色细胞，并使用多聚甲醛化学固定细胞和净化样品。
CFC提供流式细胞术的功能，包括批量采样和多参数互相关，用于抗原特异性记忆反应的分析。使用CFC的研究者能够表型表征用测试抗原和表型亚型（例如，CD4 + 或CD8 + T细胞）培养的细胞，确定高于背景水平的产生频率的细胞因子。
与ELISPOT和Luminex方法相反，CFC可以关联来自特定的，表型 - 表征的细胞的多种细胞因子的产生。 ...
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