Calcein-AM和Annexin V在流式細(xì)胞術(shù)檢測(cè)早期

1、 避光染色15rain后上機(jī)檢測(cè);誘導(dǎo)前未染色組用PBS洗2次后于Binding Buffer(140mmol/L NaCl,2.5mmol/L CaCl2,10mmoL/L HEPESNaOH,pH 7.4中重懸至106/ml,取100p.1細(xì)胞懸液加入Annexin V(10斗g/m1染色30min后再加400山Bind.ingBuffer上機(jī)檢測(cè)單熒光。另取100p.1細(xì)胞懸液加入Annexin V(10pg/m1和PI(1斗g/IIll,室溫避光反應(yīng)30min后上機(jī)檢測(cè)。1.6統(tǒng)計(jì)分析細(xì)胞凋亡的檢測(cè)數(shù)據(jù)用t檢驗(yàn)。2結(jié)果2.1單參數(shù)流式細(xì)胞儀測(cè)定結(jié)果分析用FACSort 流式細(xì)胞儀,以參

2、數(shù)熒光1高度(FLl一H對(duì)數(shù)放大檢測(cè)FITC-Annexin V和Calcein熒光強(qiáng)度。為避免PI熒光對(duì)FLl的影響,此階段實(shí)驗(yàn)均采取單標(biāo)記,不染PI。用FLl一H和細(xì)胞數(shù)的直方圖來分析凋亡率。圖l中a為單標(biāo)記Calcein-AM的對(duì)照;b是單標(biāo)記CalceinAM并經(jīng)uV誘導(dǎo)后再培養(yǎng)4h的細(xì)胞,可見明顯的“凋亡峰”出現(xiàn);c是單標(biāo)記Annexin V的對(duì)照細(xì)胞;d是uV誘導(dǎo)后培養(yǎng)4h的單標(biāo)記 AnnexinV細(xì)胞,也可見“凋亡峰”出現(xiàn),但兩者位置相反。 120詈80毒40100101102103104Annexin Vc.對(duì)照120差80蠹40Calceinb.uv誘導(dǎo)10010110210

3、3104Annexin Vd.UV誘導(dǎo)CPT誘導(dǎo)后的細(xì)胞用Calcein檢測(cè),在加藥后2h其FLl-H與對(duì)照相比差異顯著(P<0.05;用An.nexinV檢測(cè)時(shí)加藥后3h與對(duì)照相比差異才具顯著性意義。uV誘導(dǎo)的細(xì)胞用Calcein檢測(cè),照射后1h差異即顯著,而用Annexin V檢測(cè)時(shí)此時(shí)間點(diǎn)在照射后2h。參見表1和圖2。囊培養(yǎng)時(shí)間(ha.CPT誘導(dǎo)后培養(yǎng)時(shí)間(hb.UV誘導(dǎo)后實(shí)線:CalceinAM檢測(cè)結(jié)果;虛線:Annexin V檢測(cè)結(jié)果。圖2CPT和uV誘導(dǎo)不同時(shí)間點(diǎn)下Calcein.AM和An-nexinV檢測(cè)凋亡結(jié)果(n=8,面±s比較2.2雙參數(shù)流式細(xì)胞分析結(jié)果為

4、了區(qū)分壞死和凋亡細(xì)胞,在前述Calcein單染的基礎(chǔ)上加染PI進(jìn)行雙參數(shù)分析。結(jié)果見圖3。R2:正常細(xì)胞R3:早期凋亡細(xì)胞R4:晚期凋亡和壞死細(xì)胞M1:凋亡細(xì)胞峰。圖3雙參數(shù)(Calcein.AM加PI流式分析法測(cè)定細(xì)胞凋亡圖I CalceinAM和Annexin V檢測(cè)細(xì)胞凋亡表1CPT和uV誘導(dǎo)下各時(shí)間點(diǎn)(hCalcein-AM與Annexin V檢測(cè)細(xì)胞凋亡率與對(duì)照比值(n=8,牙±s的比較3討論在細(xì)胞凋亡早期,會(huì)出現(xiàn)“膜外翻”現(xiàn)象,利用此現(xiàn)象建立的常規(guī)Annexin V凋亡檢測(cè)法,可有效地對(duì)細(xì)胞凋亡進(jìn)行定量檢測(cè),并可區(qū)分壞死細(xì)鷥 Calcein-AM和Annexin V在流式

5、細(xì)胞術(shù)檢測(cè)早期細(xì)胞凋亡的敏感性比較作者:王佳, 馮永東, 陶德定, 李小蘭, 肖薇, 劉雙又, 余從年, 龔建平, WANG Jia, FENG Yong-dong, TAO De-ding, LI Xiao-lan, XIAO Wei, LIU Shuang-you, YUCong-nian, GONG Jian-ping作者單位:華中科技大學(xué)同濟(jì)醫(yī)學(xué)院分子醫(yī)學(xué)中心,武漢,430030刊名: 臨床檢驗(yàn)雜志英文刊名:CHINESE JOURNAL OF CLINICAL LABORATORY SCIENCE年,卷(期:2006,24(3參考文獻(xiàn)(6條1.Jones R A;Smail A;Wi

6、lson M R Detecting mitochondrial permeability transition by confocal imagingof intact cells pinocytically loaded with calcein外文期刊 2002(162.Martinou J C;Green M R Breaking the mitochondrial barrier 20013.Valeria Petronilli;Giovanni Miotto;Marcella Canton Transient and long-lasting opening of the mito

7、chondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence外文期刊 19994.Poncet D;Boya P;Metivier D Cytofluorometric quantitation of apoptosis-driven inner mitochondrial membrane permeabilization外文期刊 2003(55.Koopman G;Reutelingsperger C P;Kujjten G A Annexin V for flow cytometric detection of phosphatidylsrine expression on B cells undergoing apoptosis 19946.Vermes I;Haanen C;Steffens-Nakken H A novel assay for apoptosis.Flow cytom