Flow+cytometry+detect+the+T+cell+subtype....ppt

1、Detection of T cell subtypes by Flow Cytometry,2,BD公司流式细胞仪,FACSCalibur,LSRII,FACSCanto,FACSCount,FACSVantage&DiVa,FACSAria,3,What is Flow Cytometry?,Principals and Applications,FLOW CYTOMETRY,FLOW,=FLUID,CYTO,=CELLS,METRY,=MEASUREMENT,Definition,Flow Cytometry * Measuring properties of cells in flow

2、 Flow sorting * Sorting (separating) cells based on properties measuring in flow * Also called Fluorescence-Activated Cell Sorting (FACS),8,FSC and SSC,Forward Scatter (FSC) -Approximate cell size & Side Orthogonal Scatter (SSC)- Cell complexity or granularity Scatter are used for preliminary identi

3、fication of cells. In a peripheral blood sample, lymphocyte, monocyte and granulocyte populations can be defined on the basis of forward and side scatter. Forward and side scatter are used to exclude debris and dead cells.,9,FSC与SSC检测器,10,FSC and SSC,11,FSC and SSC are used for preliminary identific

4、ation of cells.,14,15,Fluoresence,Fluorescent labeling allows investigation of cell structure and function. Cell autofluorescence is generated by labeling cell structures with fluorescent dyes. FACS collect fluorescence signals in one to several channels corresponding to different laser excitation a

5、nd fluorescence emission wavelength. Immunofluorescence, the most widely used application, involves the staining of cells with antibodies conjugated to fluorescent dyes such as fluorescein and phycoerythrin. often used to label molecules on the cell surface, but antibodies can be directed at targets

6、 in cytoplasm.,16,FSC and SSC are used for preliminary identification of cells.,CD8,Step 1 Direct immunofluorescent Staining Step 2 Flow cytometry analysis,Content,Materials,heparinized peripheral blood Eppendorf tubes PBS Red blood cell lysing solution Anti-mouse CD3/FITC Anti-mouse CD4/PE Anti-mou

7、se CD8/PE-CY5 Flow cytometry machine FACSCalibur/ LSRII,20,Fluorescence emission wavelengths,fluorescein isothiocyanate (FITC), (530 nm), phycoerythrin (PE), (575 nm) phycoerythrincyanine 5 tandem complex:PE-CY5 (670 nm),21,流式细胞述检测T细胞亚群,每个实验室2只小鼠，2组同学用外周血，3组同学用脾脏，3组同学用胸腺 1. 全血100 l 孵抗体裂红细胞检测 2. 取脾脏或

8、胸腺制备细胞悬液裂解红细胞细胞计数、调细胞密度（5106/ml） 100 l 细胞悬液孵抗体检测,22,外周血T细胞亚群检测,1. 取小鼠眼眶静脉血（摘眼球），肝素抗凝，取100l全血加入至流式测定管中，加入不同荧光素标记的抗小鼠CD3,CD4,CD8抗体10-20l混匀，室温避光孵育20min； 加入红细胞裂解液3ml，混匀，放置7min裂解红细胞，1000rpm离心5min, 去除上清，加入PBS 3ml 1000rpm离心5min洗涤，去除上清， 再加入PBS 0.4 ml，流式细胞仪检测阳性细胞百分率。,23,小鼠脾脏或胸腺中T细胞亚群的检测（1）,采血后的小鼠，颈椎脱臼处死，放入70

9、% 的酒精浸泡消毒2min。 脾脏摘取： 将小鼠侧身（左侧腹部朝上）置入瓶皿中，剪开腹部皮肤，透过腹膜可见到脾脏，然后剪开腹膜，取出脾脏，放入培养皿中，一个脾脏剪两半，分两组使用。 胸腺摘取： 小鼠脾脏取出后交给另一组同学，剪开胸腔，摘取胸腺。,24,小鼠脾脏或胸腺中T细胞亚群的检测（2）,细胞悬液制备： 将胸腺或脾脏放入培养皿中，立即加入少许培养液(约0.4ml）潤湿胸腺或脾脏； 再将脏器夹在两磨砂玻片之间轻轻碾磨，加入2-3ml培养液冲洗玻片； 吸取细胞悬液经200目尼龙网过虑至朔料试管中； 1000rpm离心5min, 去除上清。,25,小鼠脾脏或胸腺中T细胞亚群的检测（3）,裂解红细胞

10、，调细胞密度： 加入3 ml 红细胞裂解液，混匀，放置 7min，1000rpm离心5min, 除上清； 加3ml培养液，吹打混匀，用加样器将细胞冲入 冲入细胞计数板计数。 1000rpm离心5min，除上清，根据计数的细胞，定量加入培养液， 使细胞浓度为5106/ml；,26,孵抗体（染色）和检测： 取100l 的细胞悬液（5105 106细胞）加入至流式测定管中， 加入不同荧光素标记的抗小鼠CD3,CD4,CD8抗 体10-20l混匀，室温避光孵育20min； 加入3ml PBS, 1000rpm离心5min，去除上清，再加入0.4 ml PBS，流式细胞仪检测阳性细胞百分率。,小鼠脾脏或胸腺中T细胞亚群的检测（3）,27,细胞计数,计算公式：四个角大方格细胞数的总和/4 104 （ml）,28,流式细胞述检测对照管的设定