Laser scanning cytometry in the characterization of the proapoptotic effects of transiently transfected genes in cerebellar granule neurons.
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.


CONCLUSIONS
LSC scoring of transiently transfected CGNs offers a rapid and reliable means of characterizing proapoptotic gene effects.
Bingham
Brendan
B
Neuroscience Discovery Research, Wyeth Research, Princeton, New Jersey 08543-8000, USA. binghab@wyeth.com
Kotnis
Smita
S
McHendry-Rinde
Barbara
B
Shen
Ru
R
Wood
Andrew
A
Kennedy
Jeffrey D
JD
D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D001562:Benzimidazoles; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D053453:Caspase 9 / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology* / Q000201:enzymology / Q000473:pathology; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D047569:Laser Scanning Cytometry / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D009474:Neurons / Q000201:enzymology / Q000473:pathology*; D010641:Phenotype; D051381:Rats; D014162:Transfection
D001562:Benzimidazoles; D053148:Caspase 3; D053453:Caspase 9; C017807:bisbenzimide ethoxide trihydrochloride