Grant Jean, Bourcier Katarzyna, Wallace Stephen, Pan Dalin, Conway Alexis, Seyfert-Margolis Vicki, Wallace Paul K
University of California San Francisco, Bethesda, Maryland 20814, USA.
Cytometry B Clin Cytom. 2009 Mar;76(2):69-78. doi: 10.1002/cyto.b.20446.
FoxP3 has become a key identifier of regulatory T cells. Investigators have used a variety of antibodies and methods for detecting FoxP3 by flow cytometry. To standardize FoxP3 antibody staining for use in clinical trial samples, we tested various antibodies from different vendors, cell preparation protocols and fix/perm reagents, and cell isolation procedures. Using this optimized staining protocol, we evaluated clinical specimens from patients with multiple sclerosis (MS) or type 1 diabetes.
FoxP3 antibodies from eBioscience (236A/E7 and PCH101) and BioLegend (206D) were evaluated along with their respective methods and fix/perm reagents for preparation and staining of FoxP3 for flow cytometry. Fresh washed blood and frozen or fresh PBMC were evaluated. Upon optimization of the protocol, clinical samples (frozen PBMC) from patients with MS or type 1 diabetes and healthy control donors were evaluated with the BioLegend antibody.
Clone 206D from BioLegend yielded optimal staining and the fix/perm reagents from both eBioscience and BioLegend were comparable. Data were also comparable between cells separated by Ficoll (fresh or frozen) and washed blood samples, allowing this protocol to be applicable to different types of samples. We validated this protocol using clinical samples and saw a significant increase in FoxP3 expression in the patients with type 1 diabetes but not in the MS.
The results from this study will allow the assessment of FoxP3 by flow cytometry on samples from clinical sites that are analyzed in real time on fresh blood or frozen PBMC.
FoxP3已成为调节性T细胞的关键标志物。研究人员已使用多种抗体和方法通过流式细胞术检测FoxP3。为了标准化用于临床试验样本的FoxP3抗体染色，我们测试了来自不同供应商的各种抗体、细胞制备方案、固定/破膜试剂以及细胞分离程序。使用这种优化的染色方案，我们评估了来自多发性硬化症（MS）或1型糖尿病患者的临床标本。
评估了eBioscience公司（236A/E7和PCH101）和BioLegend公司（206D）的FoxP3抗体，以及它们各自用于流式细胞术检测FoxP3的制备和染色方法及固定/破膜试剂。对新鲜洗涤的血液以及冷冻或新鲜的外周血单个核细胞（PBMC）进行了评估。在优化方案后，使用BioLegend抗体对来自MS或1型糖尿病患者以及健康对照供体的临床样本（冷冻PBMC）进行了评估。
BioLegend公司的克隆206D产生了最佳染色效果，eBioscience公司和BioLegend公司的固定/破膜试剂相当。通过Ficoll分离的细胞（新鲜或冷冻）和洗涤后的血液样本之间的数据也具有可比性，这使得该方案适用于不同类型的样本。我们使用临床样本验证了该方案，发现1型糖尿病患者的FoxP3表达显著增加，而MS患者则没有。
本研究结果将有助于通过流式细胞术对来自临床站点的样本进行FoxP3评估，这些样本可在新鲜血液或冷冻PBMC上进行实时分析。
