Detection of localized caspase activity in early apoptotic cells by laser scanning cytometry
Detection of localized caspase activity in early apoptotic cells by laser scanning cytometry
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DOI：
10.1002/cyto.10052
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发表时间：
2002-02-01
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影响因子：
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通讯作者：
Packard, BZ
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作者：
Telford, WG;Komoriya, A;Packard, BZ
背景：半胱天冬酶激活是细胞凋亡发生的关键早期步骤。细胞可渗透的荧光半胱天冬酶底物已被证明在通过流式细胞术检测半胱天冬酶活化方面是有价值的。然而，早期低水平的胱天蛋白酶激活的检测已经很难使用传统的面积或峰值荧光分析流式细胞术，尽管这些细胞的明显存在，通过显微镜观察。方法：以PhiPhiLux-G（1）DZ半胱氨酸蛋白酶3/7为底物，结合DNA染料排斥和膜联蛋白V（annexin V）结合技术，采用传统流式细胞术和激光扫描细胞术（LSC）检测小鼠胸腺瘤细胞EL 4中凋亡的几个阶段。单个细胞中最大像素亮度的LSC分析表明，流式细胞仪或LSC积分分析无法检测到中间半胱天冬酶-低亚群。半胱天冬酶活性的LSC分析，然后进行使用较大的UMR-106大鼠骨肉瘤细胞系，以确定这种明显的早期半胱天冬酶活性可以与本地化，点状半胱天冬酶活性通过microscopics.Results观察：凋亡的EL 4细胞中发现的半胱天冬酶低亚群也观察到UMR-106细胞。搬迁到细胞低荧光由于caspase活性和随后的检查，显微镜下显示，这些后者的细胞确实显示点状，高度本地化的caspase激活灶，可能代表一个早期阶段的caspase activation.Conclusions：细胞低水平，本地化的caspase表达可以检测到使用最大像素分析LSC。这种方法允许细胞凋亡激活的早期步骤被解析以用于进一步分析。Cytometry 47：81-88，2002. (C)2002 Wiley-Liss，Inc.
Background: Caspase activation is a critical early step in the onset of apoptosis. Cell-permeable fluorogenic caspase substrates have proven valuable in detecting caspase activation by flow cytometry. Nevertheless, detection of early low-level caspase activation has been difficult using conventional area or peak fluorescence analysis by flow cytometry, despite the apparent presence of these cells as observed by microscopy. We describe a method utilizing maximum fluorescence pixel analysis by laser scanning cytometry (LSC) to detect early apoptotic cells.Methods: The PhiPhiLux-G(1)DZ caspase 3/7 substrate was used in combination with DNA dye exclusion and annexin V binding to identify several stages of apoptosis in EL4 murine thymoma cells by both traditional flow and LSC. LSC analysis of maximum pixel brightness in individual cells demonstrated an intermediate caspase-low subpopulation not detectable by flow or LSC integral analysis. LSC analysis of caspase activity was then carried out using the larger UMR-106 rat osteosarcoma cell line to determine if this apparent early caspase activity could be correlated with localized, punctate caspase activity observed by microscopy.Results: The caspase-low subpopulation found in apoptotic EL4 cells was also observable in UMR-106 cells. Relocation to cells with low fluorescence due to caspase activity and subsequent examination by microscopy demonstrated that these latter cells indeed show punctate, highly localized caspase activation foci that might represent an early stage in caspase activation.Conclusions: Cells with low-level, localized caspase expression can be detected using maximum pixel analysis by LSC. This methodology allows an early step of apoptotic activation to be resolved for further analysis. Cytometry 47:81-88, 2002. (C) 2002 Wiley-Liss, Inc.