Journal Article

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

YAN, GE, EFFERTH, THOMAS

2018

Overview

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

• Publisher: International Institute of Anticancer Research
• Subject: Adherent cells / Annexin A5 - chemistry / Annexin V / Apoptosis / Apoptosis - drug effects / Apoptosis - physiology / Binding / Biological Assay - methods / Buffers / Cell Adhesion - drug effects / Cell Adhesion - physiology / Cell Culture Techniques / Cell death / Cell Line, Tumor / Cell Separation - methods / Conflicts of interest / Damage / Flow Cytometry / Homeostasis / Humans / Integrity / Iodides / Membranes / Methods / Permeability / Plasma / Propidium - chemistry / Propidium iodide / Rubber / Staining / Staining and Labeling - methods / Trypsin / Trypsin - pharmacology