1、Detection of T cell subtypes by Flow Cytometry2BD公司流式細(xì)胞儀公司流式細(xì)胞儀FACSCaliburLSRIIFACSCantoFACSCountFACSVantage&DiVaFACSAria3What is Flow Cytometry?Principals and ApplicationsFLOW CYTOMETRYFLOW=FLUIDCYTO=CELLSMETRY=MEASUREMENTDefinitionFlow Cytometry * Measuring properties of cells in flowFlow sorting

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

3、ls. vIn 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.9FSC與SSC檢測(cè)器 10FSC and SSC11FSC and SSC are used for preliminary identification of cells. 14

4、CD8 Step 1 Direct immunofluorescent Staining Step 2 Flow cytometry analysisContentContentMaterials Mouse spleen Eppendorf tubes PBS Red blood cell lysing solution Anti-mouse CD3/FITC Anti-mouse CD4/PE Anti-mouse CD8/APC Flow cytometry machine FACSCalibur/ LSRII18Fluorescence emission wavelengths flu

5、orescein isothiocyanate (FITC), (530 nm), phycoerythrin (PE), (575 nm) phycoerythrincyanine 5 tandem complex:PE-CY5 (670 nm)19流式細(xì)胞術(shù)檢測(cè)小鼠脾臟流式細(xì)胞術(shù)檢測(cè)小鼠脾臟T T細(xì)胞亞群細(xì)胞亞群20基本步驟基本步驟小鼠脾細(xì)胞的獲得小鼠脾細(xì)胞的獲得裂解紅細(xì)胞、并細(xì)胞計(jì)數(shù)裂解紅細(xì)胞、并細(xì)胞計(jì)數(shù)熒光抗體染色熒光抗體染色流式細(xì)胞術(shù)檢測(cè)流式細(xì)胞術(shù)檢測(cè)T細(xì)胞細(xì)胞21小鼠脾臟小鼠脾臟T細(xì)胞亞群的檢測(cè)（細(xì)胞亞群的檢測(cè)（1）v頸椎脫臼處死小鼠，放入頸椎脫臼處死小鼠，放入70% 70% 的酒

6、精浸泡消毒的酒精浸泡消毒1min1min。v脾臟摘取：脾臟摘?。?將小鼠側(cè)身（左側(cè)腹部朝上）置入瓶皿中，剪開將小鼠側(cè)身（左側(cè)腹部朝上）置入瓶皿中，剪開腹部皮膚，透過(guò)腹膜可見到脾臟，然后剪開腹膜，腹部皮膚，透過(guò)腹膜可見到脾臟，然后剪開腹膜，取出脾臟，放入培養(yǎng)皿中，一個(gè)脾臟剪取出脾臟，放入培養(yǎng)皿中，一個(gè)脾臟剪5 5塊，供塊，供5 5組使用。組使用。22小鼠脾臟小鼠脾臟T細(xì)胞亞群的檢測(cè)（細(xì)胞亞群的檢測(cè)（2）1.1.細(xì)胞懸液制備：細(xì)胞懸液制備： 取一個(gè)小平皿，放入細(xì)胞篩網(wǎng)，加入約取一個(gè)小平皿，放入細(xì)胞篩網(wǎng)，加入約1ml 1640液，液，將小塊脾臟放在細(xì)胞篩網(wǎng)上，用玻璃注射器芯碾磨，將小塊脾臟放在細(xì)胞篩

7、網(wǎng)上，用玻璃注射器芯碾磨，邊磨邊加入邊磨邊加入2ml 1640培養(yǎng)液沖洗，收集細(xì)胞懸液至培養(yǎng)液沖洗，收集細(xì)胞懸液至塑料試管中塑料試管中, 1000rpm離心離心5min，去上清；，去上清； 23小鼠脾臟小鼠脾臟T細(xì)胞亞群的檢測(cè)（細(xì)胞亞群的檢測(cè)（3）2. 裂解紅細(xì)胞，調(diào)細(xì)胞密度裂解紅細(xì)胞，調(diào)細(xì)胞密度v向細(xì)胞沉淀中加入向細(xì)胞沉淀中加入300l 1640液混勻重懸細(xì)胞；液混勻重懸細(xì)胞；v 吸取吸取100l加入加入3ml預(yù)溫的紅細(xì)胞裂解液，混勻，放置預(yù)溫的紅細(xì)胞裂解液，混勻，放置7min，1000rpm離心離心5min, 去除上清。去除上清。v加入適量的加入適量的1640 培養(yǎng)液培養(yǎng)液(約約200ul

8、)，調(diào)整細(xì)胞濃度為，調(diào)整細(xì)胞濃度為1107/ml；吸?。晃?0ul加入到流式管。加入到流式管。v剩下的剩下的150ul用于細(xì)胞記數(shù)用于細(xì)胞記數(shù): 進(jìn)行計(jì)數(shù)加入進(jìn)行計(jì)數(shù)加入1640 培養(yǎng)液培養(yǎng)液5ml，吹打混勻，用加樣器吸出吹打混勻，用加樣器吸出10l細(xì)胞懸液，沖入細(xì)胞計(jì)數(shù)板細(xì)胞懸液，沖入細(xì)胞計(jì)數(shù)板計(jì)數(shù)。計(jì)數(shù)。243. 孵抗體（染色）和檢測(cè)：孵抗體（染色）和檢測(cè)：v 取取50l 濃度為濃度為1107/ml的細(xì)胞懸液（的細(xì)胞懸液（5105細(xì)細(xì)胞）加入至流式測(cè)定管中，加入不同熒光素標(biāo)記胞）加入至流式測(cè)定管中，加入不同熒光素標(biāo)記的抗小鼠的抗小鼠CD3、CD4或或CD3/CD4抗體抗體10l，混勻，混

9、勻，室溫避光孵育室溫避光孵育20min；v加入加入3ml 含含2% 小牛血清的小牛血清的PBS(簡(jiǎn)稱簡(jiǎn)稱PBS), 1000rpm離心離心5min，去除上清，再加入，去除上清，再加入0.4 ml PBS，流式細(xì)胞儀檢測(cè)陽(yáng)性細(xì)胞百分率。，流式細(xì)胞儀檢測(cè)陽(yáng)性細(xì)胞百分率。 小鼠脾臟小鼠脾臟T細(xì)胞亞群的檢測(cè)（細(xì)胞亞群的檢測(cè)（3）25細(xì)胞計(jì)數(shù)細(xì)胞計(jì)數(shù)計(jì)算公式：四個(gè)角大方格細(xì)胞數(shù)的總和計(jì)算公式：四個(gè)角大方格細(xì)胞數(shù)的總和/4 104 （ml）26細(xì)胞計(jì)數(shù)要點(diǎn)：1.進(jìn)行細(xì)胞計(jì)數(shù)時(shí)，要求懸液中細(xì)胞數(shù)目不低于104個(gè)/ml，如果細(xì)胞數(shù)目很少要進(jìn)行離心再懸浮于少量培養(yǎng)液中；2.要求細(xì)胞懸液中的細(xì)胞分散良好，否則影響計(jì)數(shù)準(zhǔn)確性。3.取樣計(jì)數(shù)前，應(yīng)充分混勻細(xì)胞懸液，尤其是多次取樣計(jì)數(shù)時(shí)更要注意每次取樣都要混勻，以求計(jì)數(shù)準(zhǔn)確；4.數(shù)細(xì)胞的原則是只數(shù)完整的細(xì)胞，若細(xì)胞聚集成團(tuán)時(shí)，只按照一個(gè)細(xì)胞計(jì)算。如果細(xì)胞壓在格線上時(shí)，則只計(jì)上線，不計(jì)下線，只計(jì)右線，不計(jì)左線。5.將細(xì)胞懸液滴入計(jì)數(shù)板：將待測(cè)細(xì)胞懸液吹均勻，然后吸取少量懸液沿蓋片邊緣緩緩滴入，要保證蓋片下充滿懸液，注意蓋片下不要有氣泡，也不能讓懸液流入旁邊槽中。否則要重新計(jì)數(shù)。27細(xì)胞數(shù)細(xì)胞數(shù)/mL原液原液=（4大方格細(xì)胞數(shù)之和大方格細(xì)胞數(shù)之和/4）104稀釋倍數(shù)稀釋倍數(shù)公式中乘以公式中乘以104