Novel method for cell debris removal in the flow cytometric cell cycle analysis using carboxy-fluorescein diacetate succinimidyl ester.
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 gamma 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.
Terho
Perttu
P
Department of Medical Microbiology, University of Turku, Finland. perttu.terho@utu.fi
Lassila
Olli
O
D000818:Animals; D002453:Cell Cycle; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002645:Chickens / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D016393:Lymphoma, B-Cell; D013194:Staining and Labeling / Q000379:methods*; D013388:Succinimides
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013388:Succinimides