Cell harvesting method influences results of apoptosis analysis by annexin V staining

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...

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Bibliographic Details
Published in:Anticancer research 2013-08, Vol.33 (8), p.3201-3204
Main Authors: Bundscherer, Annika, Malsy, Manuela, Lange, Renate, Hofmann, Petra, Metterlein, Thomas, Graf, Bernhard M, Gruber, Michael
Format: Article
Language:English
Subjects:
Adolescent
Animals
Annexin A5 - metabolism
Apoptosis
Cell Line, Tumor
Cell Separation - methods
Female
Humans
Mice
Staining and Labeling
Trypsin - metabolism
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Summary: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. 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. 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. To avoid substantial experimental bias caused by membrane damage, we recommend pre-testing of different harvesting methods before performing apoptosis analysis.
ISSN:1791-7530