T亚群检测.docVIP

Detection of T lymphocyte subgroups in Peripheral Blood by the SAP SAP法检测人外周血T细胞亚群 【Materials】 该试剂盒内容主要试剂均为美国ZYMED公司产品。 ① 防脱片剂（Adhesion solution） ② 固定剂 (Fixed agent) ③ 小鼠抗人CD3单抗 (mouse anti-human CD3 McAb) 小鼠抗人CD4单抗 (mouse anti-human CD4 McAb) 小鼠抗人CD8单抗 (mouse anti-human CD8 McAb) ④ 生物素标记羊抗小鼠IgG (IgG/Bio) Goat anti-mouse IgG labeled with biotin ⑤ 碱性磷酸酶标记链霉卵白素(S-A/AP)（抗生物素蛋白链霉菌素） strepto-avidin labeled with alkali phosphatase ⑥ A液：40×底物增强剂 (40×substrate enhanced solution) B液：40×底物液(40×substrate solution) C液：20×底物缓冲液(20×substrate buffer) ⑦ PBS缓冲液 (sodium phosphate-buffered saline) 【Methods】 1. Sample preparation (标本制备) Separation of mononuclear cells suspension from whole peripheral blood. 制备外周血单个核细胞悬液。 Glass slide should be pre-treated by smearing 5~10μl adhesion solution on it and dry it before adding the PBMC suspension. 涂片前，取防脱片剂5~10μl均匀涂于洁净玻片上，待干后制备细胞涂片，以防掉片。 Adding 30μl PBMC suspension onto the treated slide. Waiting 2min for the sinking and adhering of the cells to the slide. Erasured the liquid and dry it by blowing fleetly in a short time or roasting at 37℃ for 1 hour. 取30μl细胞悬液滴于载玻片上，静置2min, 使细胞下沉粘附于玻片上，小心吸去液体，快速吹干或37℃1h烘干。 2. Sample Dye （标本染色） Limit the cell sample area using a marker pen. Add 50μl fixation solution in the circle and wait for 2~3min. Wash the slide by PBS buffer repeatedly. Blot up the liquid outside the marked area and the back of the slide 先用记号笔在，充分干燥（室温2小时以上或过夜）的细胞涂片标本外画圈，然后滴加固定液50μl，室温静置2~3min，PBS淋洗，擦干玻片背面及细胞外的PBS液体。 **All rest procedures are taking in a wet box. 以下反应均需在湿盒中进行。 Add 10~30μl mouse anti-human CD3 (CD4 or CD8) monoclonal antibodies into the cell area and incubate the slide at 37℃ for 30min. Wash it by PBS for 3 times. 分别滴加适量CD3、CD4、CD8单抗 (10~30μl)，37℃孵育30min，PBS淋洗3次。 Add 10~30μl goat anti-mouse IgG labeled with biotin and incubate the slide at 37℃ for 30min. Wash it by PBS for 3 times. 滴加适量生物素标记羊抗小鼠IgG (10~30μl)，37℃孵育30min，PBS淋洗3次。 Add 10~30μl strepto-avidin labeled with alkali phosphatase and incubate th