title: Screening assay for oxidative stress in a feline astrocyte cell line, G355-5.
abstract: An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of the Central Nervous System (CNS). Astrocytes help maintain a homeostatic environment for neurons as well as protecting neurons from Reactive Oxygen Species (ROS). CM-H2DCFDA is a cell-permeable indicator for the presence of ROS. CM-H(2)DCFDA enters the cell as a non-fluorescent compound, and becomes fluorescent after cellular esterases remove the acetate groups, and the compound is oxidized. The number of cells, measured by flow cytometry, that are found to be green fluorescing is an indication of the number of cells that are in an oxidative state. CM-H(2)DCFDA is susceptible to oxidation by a large number of different ROS. This lack of specificity, regarding which ROS can oxidize CM-H(2)DCFDA, makes this compound a valuable regent for use in the early stages of a pathogenesis investigation, as this assay can be used to screen for an oxidative cellular environment regardless of which oxygen radical or combination of ROS are responsible for the cellular conditions. Once it has been established that ROS are present by oxidation of CM-H(2)DCFDA, then additional experiments can be performed to determine which ROS or combination of ROSs are involved in the particular pathogenesis process. The results of this study demonstrate that with the addition of hydrogen peroxide an increase in CM-H(2)DCFDA fluorescence was detected relative to the saline controls, indicating that this assay is a valuable test for detecting an oxidative environment within G355-5 cells, a feline astrocyte cell line.
authors.affiliation: College of Veterinary Medicine, Western University of Health Sciences, USA.
authors.forename: Maria Pia
authors.initials: MP
authors.lastname: Testa
authors.forename: Omar
authors.initials: O
authors.lastname: Alvarado
authors.forename: Andrea
authors.initials: A
authors.lastname: Wournell
authors.forename: Jonathan
authors.initials: J
authors.lastname: Lee
authors.forename: Frederick T
authors.initials: FT
authors.lastname: Guilford
authors.forename: Steven H
authors.initials: SH
authors.lastname: Henriksen
authors.forename: Tom R
authors.initials: TR
authors.lastname: Phillips
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002415:Cats; D002460:Cell Line; D005434:Flow Cytometry; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; C037631:2',7'-dichlorofluorescein; D006861:Hydrogen Peroxide