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

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.