Flow cytometry as a method for studying effects of stressors on primary rat neurons

Abstract

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.

PTEN, Akt, and GSK3β signalling in rat primary cortical neuronal cultures following tumor necrosis factor‐α and trans‐4‐hydroxy‐2‐nonenal treatments

“…The 24-hr incubation gave the best and most reproducible separation of these different populations. Therefore, in this study, we chose a single time point for changes of protein phosphorylation that correlated with our previous toxicity data (Behbahani et al, 2005).…”

“…The methodology for this in rat primary neuronal cells was described in detail in a recent paper (Behbahani et al, 2005). In brief, primary neurons were treated with 6.84 mM EDTA (Sigma, St. Louis, MO) and 0.05% trypsin.…”

“…The phosphorylation of Ser 380 PTEN, Ser 473 Akt, and Ser 9 GSK3b after treatment with TNFa and HNE was determined by Western blot with specific antibodies that recognize phosphorylated forms of these proteins. To evaluate the effects of TNFa-and HNE-induced toxicity on these primary cultures, we determined populations of healthy, apoptotic, and necrotic cells by using flow cytometry (Behbahani et al, 2005).…”

G-Protein-Coupled Receptor 30 Mediates Rapid Neuroprotective Effects of Estrogen via Depression of NR2B-Containing NMDA Receptors

“…Apoptosis analyses were performed as described previously (Behbahani et al, 2005). Apoptosis and cell viability were measured using annexin V-FITC (Assay Designs) and propidium iodide (PI) double staining.…”

Efeitos do estresse pré-natal sobre a atividade de linfócitos de uma prole de camundongos

“…Assim, utilizou-se o fluorocromo PI (iodeto de propídeo) o qual indica perda de seletividade de membrana e conseqüentemente, células inviáveis por necrose. Usou-se, também, a anexina-FITC, um marcador específico de fosfatidil-serina, proteína fisiologicamente expressa na parte intracelular da membrana que ao ser externalizada iniciando o processo apoptótico mediado pela cascata de eventos das caspases (BEHBAHANI et al, 2005).…”