Cell Harvesting Methods Affect Cellular Integrity of Adherent Cells During Apoptosis Detection.

BACKGROUND/AIM: Annexin V and propidium iodide (PI) dual staining is commonly applied in bioscience as a method to detect apoptosis. However, excessive handling of adherent cells may interfere with the integrity of plasma membrane and hence impede the accuracy of this method. Here, we exploited PI uptake as an indicator of cell integrity and investigated how cell harvesting methods and solutions involved in common apoptosis detection techniques affected measurement results.

MATERIALS AND METHODS

Different cell harvesting techniques, staining with PI and flow cytometry were performed.

RESULTS

Non-fixed scrapped cells revealed significantly higher fractions of PI-positive staining compared to non-fixed trypsinized cells. In the case of harvesting cells by scrapping, samples stained in binding buffer (68.30±3.55%) showed consistently higher PI-positive staining than samples stained in PBS (36.37±5.90%) in a significant manner (p=0.015).

CONCLUSION

Enzymatic harvesting using 0.25% trypsin instead of mechanical harvesting by rubber scraper caused less damage of cell integrity. Furthermore, the binding buffer used in the apoptosis detection protocol aggravated the existing plasma membrane damage caused by the rubber scraper.