Analysis of murine lymphocyte subpopulations by dual-color flow cytometry: technical considerations and specificities of monoclonal antibodies directed against surface markers.
Analysis of murine lymphocyte subpopulations by dual-color flow cytometry: technical considerations and specificities of monoclonal antibodies directed against surface markers.
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通过双色流式细胞术分析小鼠淋巴细胞亚群：针对表面标记的单克隆抗体的技术考虑和特异性。
DOI：
10.1016/0022-4804(88)90040-6
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发表时间：
1988
期刊：
影响因子：
--
通讯作者：
Gadd,MA
中科院分区：
文献类型：
--
作者：
Hansbrough,JF;Soderberg,C;FieldJr,TO;Swisher,S;Brahme,J;Zapata-Sirvent,RL;Tonks,M;Gadd,MA
我们使用市售的单克隆抗体对来自胸腺、脾脏、淋巴结和外周血的小鼠淋巴细胞进行了详细的表型分析，每种单克隆抗体均具有膜表面标志物特异性和双色流式细胞术。红细胞裂解技术用于淋巴细胞制备，从而避免梯度技术的固有困难，例如异常大小细胞的损失。这些研究表明，每种单克隆抗体的特异性必须仔细确定;例如，Lyt-1单克隆抗体，经常用于识别辅助/诱导T细胞，也与抑制/细胞毒性（Lyt-2+）细胞反应;辅助/诱导细胞更好地研究了最近可用的单克隆抗体，L3 T4。来自不同组织的细胞可能不仅在表面标记物的存在方面存在很大差异，而且在每种标记物的表面密度方面也存在很大差异;可以使用适当的分析软件来研究和定量该密度。我们还表明，更大和更多的颗粒淋巴细胞似乎是丰富的表面Ia抗原，表明这些细胞可能是激活或调节亚群;这些大，Ia+ T细胞将失去分析，如果标准，窄门设置用于分析前向和侧向散射特性或细胞分选。
We performed detailed phenotypic analysis of murine lymphocytes from thymus, spleen, lymph node, and peripheral blood using commercially available monoclonal antibodies, each with specifities for membrane surface markers and dual-color flow cytometry. Erythrocyte lysis techniques were utilized for lymphocyte preparation so that inherent difficulties with gradient techniques would be avoided, such as the potential for loss of abnormally sized cells. These studies demonstrated that the specifities of each monoclonal must be carefully determined; for example, the Lyt-1 monoclonal, frequently utilized to identify helper/inducer T cells, also reacts with suppressor/cytotoxic (Lyt-2+) cells; helper/inducer cells are better studied with a more recently available monoclonal, L3T4. Cells from different tissues may differ greatly not only in the presence of surface markers, but also in the surface density of each marker; this density can be studied and quantitated using appropriate analytic software. We also show that larger and more granular lymphocytes appear to be enriched for surface Ia antigen, indicating that these cells may be activated or regulatory subsets; these large, Ia+T-cells will be lost from analysis if standard, narrow gate settings are used for analyzing forward and side-scatter characteristics or for cell sorting.