Novel method for cell debris removal in the flow cytometric cell cycle analysis using carboxy-fluorescein diacetate succinimidyl ester

Abstract

Background: Cell cycle analysis with flow cytometry using propidium iodide (PI) can be difficult in some cases because of the cell debris. Here, we introduce debris removal using intranuclear protein staining (DRIPS), a novel method for separating intact nuclei and cell debris to different populations using carboxy-fluorescein diacetate succinimidyl ester (CFSE). Methods: To study the apoptosis-sensitivity, chicken DT40 B cell lymphoma cell line was γ irradiated. After the irradiation, the cells were incubated up to 8 h and the stages of the cell cycle were followed with flow cytometry. Results: CFSE staining, done simultaneously with PI, stained the cell debris brighter than intact nuclei and could be excluded from the histogram with a simple gating procedure. The method is reliable and reproducible and can be executed within 15 min. Conclusions: DRIPS-method greatly enhances the analysis of difficult cell cycle samples. © 2006 International Society for Analytical Cytology.