细胞收获方法会影响 Annexin V 染色法分析细胞凋亡的结果。
Cell harvesting method influences results of apoptosis analysis by annexin V staining.
机构信息
Department of Anesthesiology, University of Regensburg, Franz Josef Strauß Allee 11, 93053 Regensburg, Germany.
出版信息
Anticancer Res. 2013 Aug;33(8):3201-4.
BACKGROUND
Annexin V staining is a common tool in apoptosis analysis. However, in adherently growing cell lines, substantial experimental bias could be introduced by membrane damage during the harvesting process. We investigated the influence of three different harvesting methods on the cell membrane integrity of six malignant cell lines.
MATERIALS AND METHODS
Six malignant cell lines were detached enzymatically by standard trypsinization or mechanically by scraping or wash-down by water jet. Membrane damage was measured by annexin V staining.
RESULTS
Three out of six cell lines (Mel-Ho, SW480 and PaTu 8988t) were not susceptible to membrane damage long the mothods used here. In HT 29, PANC 1 and A-673 cell lines, a high percentage of cells were stained positively for annexin V after mechanical detachment. These cells would wrongly be declared apoptotic cells.
CONCLUSION
To avoid substantial experimental bias caused by membrane damage, we recommend pre-testing of different harvesting methods before performing apoptosis analysis.
背景
膜联蛋白 V 染色是凋亡分析的常用工具。然而，在贴壁生长的细胞系中，在收获过程中细胞膜可能会受到损伤，从而引入大量的实验偏差。我们研究了三种不同的收获方法对六种恶性细胞系细胞膜完整性的影响。
材料与方法
通过标准的胰蛋白酶消化或机械刮擦或水流冲洗将六种恶性细胞系进行酶促或机械分离。通过膜联蛋白 V 染色测量细胞膜损伤。
结果
六种细胞系中的三种（Mel-Ho、SW480 和 PaTu 8988t）不易受到这里使用的方法引起的细胞膜损伤。在 HT 29、PANC 1 和 A-673 细胞系中，机械分离后，很大比例的细胞对膜联蛋白 V 呈阳性染色。这些细胞将被错误地判定为凋亡细胞。
结论
为了避免由于细胞膜损伤引起的大量实验偏差，我们建议在进行凋亡分析之前，对不同的收获方法进行预测试。
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