Quantification of sPLA2-induced early and late apoptosis changes in neuronal cell cultures using combined TUNEL and DAPI staining
Quantification of sPLA2-induced early and late apoptosis changes in neuronal cell cultures using combined TUNEL and DAPI staining
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DOI：
10.1016/j.brainresprot.2004.04.001
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发表时间：
2004-08-01
期刊：
影响因子：
--
通讯作者：
DeCoster, MA
中科院分区：
文献类型：
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作者：
Daniel, B;DeCoster, MA
末端脱氧核苷酸转移酶(TdT)dUTP缺口末端标记(TUNEL)染色被广泛用于检测神经元以及其他类型细胞的凋亡。TUNEL可能会给出假阳性结果，因为标记技术的变化以及对经历了非凋亡性DNA链断裂的细胞的染色。因此，孤立地讲，TUNEL不是细胞凋亡的特定指标。最近，我们证实了分泌的第三组磷脂酶A(2)对原代培养的大鼠皮质神经元具有明显的凋亡作用。在这里，我们描述了一种计算机辅助的方法，用于对SplA(2)-III诱导的细胞凋亡后的TUNEL阳性神经元进行量化。用4‘，6-二氨基-2-苯基吲哚(DAPI)染色将TUNEL的范围归一化为总的核含量。此外，DAPI反染色允许根据核的大小和圆度来确定核形态指标，我们称之为核面积因子。我们发现，核面积因子是细胞死亡的早期指标(在治疗后4小时显著)，而TUNEL染色在以后的时间(26小时)显著。因此，使用TUNEL和DAPI的独立染色技术相互补充，并与商用图像分析软件相结合，可用于显示早期和延迟的细胞损伤过程。(C)2004爱思唯尔B.V.保留所有权利。
The terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) stain is in wide use for measuring apoptosis in neurons, as well as in other cell types. TUNEL may give false positive results due to variations in labeling technique as well as staining of cells that have undergone non-apoptotic DNA strand breaks. Therefore, in isolation, TUNEL is not a certain indicator of apoptosis. Recently, we have demonstrated the potent apoptotic effect of secreted phospholipase A(2) from group III (sPLA(2)-III) on primary cortical neurons from rat. Here we describe a computer-assisted method for quantifying TUNEL-positive neurons after sPLA(2)-III induced apoptosis. Extent of TUNEL is normalized to total nuclear content using 4',6-diamidino-2-phenylindole (DAPI) staining. Furthermore, DAPI counterstaining allows for determination of a nuclear morphology indicator, based on nuclear size and roundness, which we call the nuclear area factor. We found that the nuclear area factor is an early indicator of cell death (significant after 4 It post treatment), while TUNEL staining is significant at later times (26 h). Thus, the independent staining techniques using TUNEL and DAPI complement each other, and with commercially available image analysis software, may be used to indicate early as well as delayed cell injury processes. (C) 2004 Elsevier B.V. All rights reserved.