The importance of Foxp3 antibody and fixation/permeabilization buffer combinations in identifying CD4+CD25+Foxp3+ regulatory T cells.
The importance of Foxp3 antibody and fixation/permeabilization buffer combinations in identifying CD4+CD25+Foxp3+ regulatory T cells.
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DOI：
10.1002/cyto.a.20815
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发表时间：
2009-12
期刊：
影响因子：
3.7
通讯作者：
Lanteri, Marion C.
中科院分区：
文献类型：
--
作者：
Law, Jacqueline P.;Hirschkorn, Dale F.;Owen, Rachel E.;Biswas, Hope H.;Norris, Philip J.;Lanteri, Marion C.
关键词：
Foxp 3是CD 4+调节性T细胞（Tcells）的关键标志物，并用于开发多参数流式细胞仪组以鉴定Tcells。实现可重复的染色和分析首先需要优化Foxp 3染色。我们提出了抗人Foxp 3抗体的PCH 101、236 A/E7、3G 3、206 D、150 D和259 D/C7克隆的比较研究，其与五种不同的固定/透化缓冲液组合使用。还比较了固定/透化处理之间的CD 25、CD 152和CD 127染色。在来自10个个体的一组PBMC中测试有希望的抗体/缓冲液组合，然后在来自4个个体的新鲜与冷冻细胞上测试。最后，比较与两种代表性抗体偶联的不同荧光染料以优化Foxp 3+与Foxp 3-事件的分离。使用两种门控策略，基于CD 127 + CD 25-“非Tcl 3”或基于同种型对照，设置Foxp 3门控。对于Foxp 3染色，使用eBioscience Foxp 3、Imgenex、BioLegend和BD Foxp 3缓冲液获得固定/透化的最佳条件。比较来自10名受试者的结果，259 D/C7、PCH 101、236 A/E7和206 D抗体产生的Foxp 3细胞水平在统计学上高于150 D和3G 3抗体（平均值分别为6.9、5.1、4.7和3.7%，相比之下，CD 4+细胞内的CD 25 + Foxp 3+事件分别为1.7和0.3%）。重要的是，基于同种型对照，用Foxp 3门观察到的一些抗体的“非特异性”可以通过将Foxp 3门设置为“非Tcl 2”来消除。与FITC相比，使用与Alexa 647偶联的PCH 101克隆，或与Alexa 488荧光染料相比，使用与PE偶联的259 D/C7克隆，观察到Foxp 3+和Foxp 3-群体的更好分离。Foxp 3染色可以是高度可变的，并且取决于抗体/缓冲液对的选择和所使用的荧光染料。选择用于设置Foxp 3门的正确群体对于避免在Foxp 3+门中包括非T细胞至关重要。本文提供的实验将有助于优化流式细胞术染色组，以定量人体中的Treg频率。
Foxp3 is a key marker for CD4+ regulatory T cells (Tregs) and was utilized in developing a multiparameter flow cytometric panel to identify Tregs. Achieving reproducible staining and analysis first required optimization of Foxp3 staining. We present a comparative study of PCH101, 236A/E7, 3G3, 206D, 150D, and 259D/C7 clones of anti-human-Foxp3 antibodies, used in combination with five different fixation/permeabilization buffers. Staining for CD25, CD152, and CD127 was also compared between fixation/permeabilization treatments. Promising antibody/buffer combinations were tested in a panel of PBMCs from 10 individuals, then on fresh versus frozen cells from four individuals. Finally, different fluorochromes coupled to two representative antibodies were compared to optimize separation of Foxp3+ from Foxp3- events. Foxp3 gates were set using two gating strategies, based on CD127+CD25- “non-Tregs” or based on isotype controls. For Foxp3 staining the best conditions for fixation/permeabilization were obtained using the eBioscience Foxp3, Imgenex, BioLegend, and BD Foxp3 buffers. Comparing results from 10 subjects, 259D/C7, PCH101, 236A/E7, and 206D antibodies yielded statistically higher levels of Foxp3 cells than 150D and 3G3 antibodies (mean=6.9, 5.1, 4.7, and 3.7% compared to 1.7, and 0.3% of CD25+Foxp3+ events within CD4+ cells, respectively). Importantly, the “non-specificity” of some antibodies observed with a Foxp3 gate based on isotype controls could be eliminated by setting the Foxp3 gate on “non-Tregs”. Better separation of Foxp3+ and Foxp3- populations was observed using the PCH101 clone coupled to Alexa647 compared to FITC, or the 259D/C7 clone coupled to PE compared to Alexa488 fluorochrome. Foxp3 staining can be highly variable and depends on the choice of antibody/buffer pair and the fluorochrome used. Selecting the correct population for setting the Foxp3 gate is critical to avoid including non-Tregs in the Foxp3+ gate. The experiments presented here will aid in optimization of flow cytometry staining panels to quantify Treg frequencies in humans.
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DOI：
10.1084/jem.20060772
发表时间：
2006-07-10
期刊：
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影响因子：
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