Quantification of neurotoxicity and identification of cellular subsets in a three-dimensional brain model.
Imaging of cells in a large intact three-dimensional tissue remains difficult. Quantification and identification of cell damage in a mixed culture system has been limited by the inability of fluorescent probes to discriminate types of cellular death and penetrate tissue more that 100 microm thick. We have investigated several probes in combination with neural cell-specific antibodies to quantify cell damage in the presence of several toxins. Acridine orange and ethidium bromide were excellent for determination of cell viability, death by necrosis, or apoptosis in thick brain tissue aggregates. Calcein and ethidium homodimer were effective on live/ dead stains, and the Syto dyes 11 and 13 worked well for quantification of all cells in the brain aggregate model. By using these combinations of dyes in conjunction with confocal microscopy, we were able to quantify neural cell damage without disrupting the three-dimensional environment.
Pulliam
L
L
Department of Laboratory Medicine, Veterans Affairs Medical Center, University of California, San Francisco 94121, USA.
Stubblebine
M
M
Hyun
W
W
D017209:Apoptosis; D001921:Brain / Q000166:cytology*; D002449:Cell Aggregation; D018929:Cell Culture Techniques / Q000379:methods; D016923:Cell Death; D002470:Cell Survival; D018690:Excitatory Amino Acid Agonists / Q000633:toxicity; D005333:Fetus; D005456:Fluorescent Dyes; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods*; D008959:Models, Neurological; D016202:N-Methylaspartate / Q000633:toxicity; D009457:Neuroglia / Q000166:cytology* / Q000187:drug effects / Q000473:pathology; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000473:pathology; D014409:Tumor Necrosis Factor-alpha / Q000633:toxicity
D018690:Excitatory Amino Acid Agonists; D005456:Fluorescent Dyes; D014409:Tumor Necrosis Factor-alpha; D016202:N-Methylaspartate