Flow cytometry as a method for studying effects of stressors on primary rat neurons.
The mechanisms associated with cell death have been an important focus for neurobiology research. In the present study, the methodology of flow cytometry was used to optimize quantification of the toxic effects of tumor necrosis factor-alpha (TNF-alpha), trans-4-hydroxy-2-nonenal (4-HNE), and aged amyloid-beta (Abeta1-42) on rat primary cortical neurons. The fluorescent dyes annexin V-FITC and propidium iodide (PI) were used to identify populations of viable, early apoptotic, necrotic and late apoptotic cells by flow cytometry. Prior to exposure, the primary cultures showed 83% cell viability. Flow cytometry following labeling of cells with a specific neuronal marker, TUJ-1, revealed 82% pure neuronal populations, whereas approximately 7% were astrocytic as shown by glial fibrillary acidic protein positivity. Exposure of primary cultures to TNF-alpha, 4-HNE, and aged Abeta1-42 gave an increased number of early apoptotic cells. We show that flow cytometry is a suitable method for quantifying effects of different stressors on neurons in primary cultures. This technique could be useful for screening and testing of pharmacological compounds relevant to neurodegenerative disorders.
Behbahani
H
H
Neurotec Department, Division of Experimental Geriatrics, Novum, KASPAC, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden. homira.behbahani@neurotec.ki.se
Rickle
A
A
Concha
H
H
Ankarcrona
M
M
Winblad
B
B
Cowburn
R F
RF
D000447:Aldehydes / Q000494:pharmacology; D016229:Amyloid beta-Peptides / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D015415:Biomarkers / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism* / Q000503:physiopathology; D004195:Disease Models, Animal; D005434:Flow Cytometry / Q000379:methods*; D009336:Necrosis / Q000139:chemically induced / Q000378:metabolism / Q000503:physiopathology; D019636:Neurodegenerative Diseases / Q000378:metabolism* / Q000503:physiopathology; D009457:Neuroglia / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009498:Neurotoxins / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D010446:Peptide Fragments / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D014404:Tubulin / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000447:Aldehydes; D016229:Amyloid beta-Peptides; D015415:Biomarkers; D009498:Neurotoxins; D010446:Peptide Fragments; D014404:Tubulin; D014409:Tumor Necrosis Factor-alpha; C075222:amyloid beta-protein (1-42); C462941:beta3 tubulin, mouse; C027576:4-hydroxy-2-nonenal