Torrero M N, Larson D, Hübner M P, Mitre E
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA.
Clin Exp Allergy. 2009 Mar;39(3):361-9. doi: 10.1111/j.1365-2222.2008.03154.x. Epub 2008 Dec 23.
Basophils are increasingly recognized as playing important roles in the immune responses of allergic diseases and helminth infections. One of the main obstacles to studying basophils has been the lack of a simple and rapid assay to measure basophil activation in mice.
The purpose of this study was to develop an assay to measure murine basophil activation.
Mouse blood cells were stained with various combinations of positive and negative markers for basophils--sorted and then assessed for basophil purity by May-Grünwald staining of cytospins. Once a flow cytometric strategy for staining basophils was determined, basophil surface expression of CD200R was assessed by multi-colour flow cytometry after stimulation of whole blood with anti-IgE, ionomycin or N-formyl MetLeuPhe (fMLP). Confirmation of basophil activation was assessed by concomitant staining of cells for intracellular IL-4. To test the ability of flow cytometric basophil CD200R measurements to assess for antigen-specific IgE-mediated activation of basophils, surface CD200R expression in response to in vitro stimulation with media alone, helminth antigen or ovalbumin was measured on basophils obtained from control mice, mice infected with helminths and mice sensitized to ovalbumin.
Using anti-IgE-FITC as a positive marker and a combination of anti-CD4-PERCP and anti-B220-PERCP as negative markers resulted in a well-separated basophil population. Additional staining with anti-CD200R-PE demonstrated that (1) basophil CD200R expression increases in response to anti-IgE, ionomycin and fMLP, (2) most CD200R-positive basophils also stain positively for IL-4 and (3) CD200R expression increases after antigen-specific activation of basophils in murine models of helminth disease and allergy.
We developed a multi-colour flow cytometry assay that measures murine basophil activation by utilizing CD200R as an activation marker. This assay is straightforward and rapid, taking approximately half a day for obtaining blood, in vitro stimulation and flow cytometric analysis.
嗜碱性粒细胞在过敏性疾病和蠕虫感染的免疫反应中所起的重要作用日益受到认可。研究嗜碱性粒细胞的主要障碍之一是缺乏一种简单快速的方法来检测小鼠嗜碱性粒细胞的活化情况。
本研究旨在开发一种检测小鼠嗜碱性粒细胞活化的方法。
用嗜碱性粒细胞的各种阳性和阴性标志物组合对小鼠血细胞进行染色——分选后通过对细胞涂片进行迈-格氏染色评估嗜碱性粒细胞纯度。一旦确定了嗜碱性粒细胞染色的流式细胞术策略，在用抗IgE、离子霉素或N-甲酰甲硫氨酰亮氨酰苯丙氨酸（fMLP）刺激全血后，通过多色流式细胞术评估嗜碱性粒细胞表面CD200R的表达。通过对细胞进行细胞内白细胞介素-4的共染色来评估嗜碱性粒细胞活化的确认情况。为了测试流式细胞术检测嗜碱性粒细胞CD200R以评估抗原特异性IgE介导的嗜碱性粒细胞活化的能力，在从对照小鼠、感染蠕虫的小鼠和对卵清蛋白致敏的小鼠获得的嗜碱性粒细胞上，测量单独用培养基、蠕虫抗原或卵清蛋白进行体外刺激后表面CD200R的表达。
使用抗IgE-异硫氰酸荧光素作为阳性标志物，抗CD4-藻红蛋白-叶绿素蛋白和抗B220-藻红蛋白-叶绿素蛋白的组合作为阴性标志物，可得到分离良好的嗜碱性粒细胞群体。用抗CD200R-藻红蛋白进行额外染色表明：（1）嗜碱性粒细胞CD200R表达在抗IgE、离子霉素和fMLP刺激下增加；（2）大多数CD200R阳性嗜碱性粒细胞白细胞介素-4染色也呈阳性；（3）在蠕虫病和过敏的小鼠模型中，嗜碱性粒细胞经抗原特异性活化后CD200R表达增加。
我们开发了一种多色流式细胞术检测方法，该方法通过利用CD200R作为活化标志物来检测小鼠嗜碱性粒细胞的活化情况。该检测方法简单快速，获取血液、体外刺激和流式细胞术分析大约需要半天时间。
