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Laser scanning cytometry in the characterization of the proapoptotic effects of transiently transfected genes in cerebellar granule neurons
Author(s) -
Bingham Brendan,
Kotnis Smita,
McHendryRinde Barbara,
Shen Ru,
Wood Andrew,
Kennedy Jeffrey D.
Publication year - 2006
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.20327
Subject(s) - transfection , propidium iodide , microbiology and biotechnology , programmed cell death , apoptosis , cytometry , biology , flow cytometry , granule (geology) , cell culture , biochemistry , genetics , paleontology
Background Low transient transfection efficiency limits the ability to characterize putative proapoptotic gene function in neurons. Laser scanning cytometry (LSC), with its high capacity, medium throughput means of collecting fluorescent emissions from cultured cells, offers an effective technology for scoring cell death in neuronal transfectants. Methods Cerebellar granule neurons (CGNs) were transfected with EGFP‐fusion constructs of Caspase‐3 and Caspase‐9 using a DNA‐calcium phosphate coprecipitation method. CGNs were fixed, permeablized, and stained with propidium iodide (PI) nuclear dye. An LSC method, based on a combination of Long Red Max Pixel, Long Red Integral, and Green Integral fluorescence parameters was validated for the scoring of apoptotic cell death in CGNs. Results In Caspase‐3 and Caspase‐9 transfected CGNs, cell death was scored both in transfectants and nontransfected culture‐mates. The cell death phenotype was found to be independent of transfection efficiency. LSC scoring of Caspase‐9 transfectants was compared with visual scoring following Hoechst 33342 staining, yielding results that were similar qualitatively, but not quantitatively, likely owing to the greater sensitivity to green fluorescence of laser scanning compared to human vision. Conclusion LSC scoring of transiently transfected CGNs offers a rapid and reliable means of characterizing proapoptotic gene effects. © 2006 International Society for Analytical Cytology