A microtiter trypan blue absorbance assay for the quantitative determination of excitotoxic neuronal injury in cell culture.
An automated method for the determination of neuronal cell death using trypan blue is described. Following various excitotoxic insults, murine mixed cortical cell cultures are stained with trypan blue (0.05%; 15 min), followed by SDS (1%) lysis. The absorbance of the dye is measured spectrophotometrically at 590 nm using a microtiter plate reader. When compared to the biochemical lactate dehydrogenase assay, no statistical difference in the calculated levels of excitotoxic neuronal cell death was noted between the assays in any given paradigm. This method is fast and reliable. It eliminates the need for cell counting, thus allowing for high volume sample analysis with a minimum of sample error. Utility of this trypan blue absorbance spectrophotometric assay is likely to extend beyond the study of excitotoxic neuronal injury and should complement existing methods for measuring neuronal viability and cytotoxicity in cell culture.
Uliasz
T F
TF
Department of Pharmacology and the Program in Neuroscience, The University of Connecticut School of Medicine, Farmington, CT 06030, USA.
Hewett
S J
SJ
D000042:Absorption; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects / Q000473:pathology; D018929:Cell Culture Techniques / Q000379:methods*; D016923:Cell Death / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured / Q000187:drug effects / Q000473:pathology; D002540:Cerebral Cortex / Q000187:drug effects / Q000473:pathology; D001330:Electronic Data Processing / Q000295:instrumentation / Q000379:methods*; D051379:Mice; D008815:Mice, Inbred Strains; D009410:Nerve Degeneration / Q000139:chemically induced* / Q000473:pathology / Q000503:physiopathology; D009474:Neurons / Q000187:drug effects / Q000473:pathology; D009498:Neurotoxins / Q000633:toxicity*; D013053:Spectrophotometry / Q000295:instrumentation / Q000379:methods*; D014343:Trypan Blue
D009498:Neurotoxins; D014343:Trypan Blue