title: Cell harvesting method influences results of apoptosis analysis by annexin V staining.
abstract: 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.
  
  
  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.
  
  
  CONCLUSIONS
  To avoid substantial experimental bias caused by membrane damage, we recommend pre-testing of different harvesting methods before performing apoptosis analysis.
authors.affiliation: Department of Anesthesiology, University of Regensburg, Franz Josef Strauß Allee 11, 93053 Regensburg, Germany. anika.bundscherer@ukr.de
authors.forename: Annika
authors.initials: A
authors.lastname: Bundscherer
authors.forename: Manuela
authors.initials: M
authors.lastname: Malsy
authors.forename: Renate
authors.initials: R
authors.lastname: Lange
authors.forename: Petra
authors.initials: P
authors.lastname: Hofmann
authors.forename: Thomas
authors.initials: T
authors.lastname: Metterlein
authors.forename: Bernhard M
authors.initials: BM
authors.lastname: Graf
authors.forename: Michael
authors.initials: M
authors.lastname: Gruber
mesh_terms: D000293:Adolescent; D000818:Animals; D017304:Annexin A5 / Q000378:metabolism*; D017209:Apoptosis; D045744:Cell Line, Tumor; D002469:Cell Separation / Q000379:methods*; D005260:Female; D006801:Humans; D051379:Mice; D013194:Staining and Labeling; D014357:Trypsin / Q000378:metabolism
chemical_list: D017304:Annexin A5; D014357:Trypsin
keywords: Apoptosis; adherent cells; annexin V staining; cancer; cell harvesting