Evaluation of glutathione-sensitive fluorescent dyes in cortical culture

Joseph S. Tauskela, Kimberley Hewitt, Li Ping Kang, Tanya Comas, Tania Gendron, Antoine Hakim, Matt Hogan, Jon Durkin, Paul Morley

Research output: Contribution to journalArticle

78 Scopus citations

Abstract

The sensitivity of six fluorophores to glutathione (GSH) was evaluated in living rat cortical neuronal/glial mixed cultures during the first 23 days in vitro (DIV). Four of the dyes require glutathione-S-transferase (GST) to form a fluorescent conjugate, potentially conferring specificity for GSH: these included t-butoxycarbonyl-Leu-Met-7-amino-4-chloromethylcoumarin (CMAC), 7-amino-4-chloromethylcoumarin (CMAC-blue), monochlorobimane (MCB), and 5-chloromethylfluorescein diacetate (CMFDA). The final two dyes examined, 2,3-naphthalenedicarboxaldehyde (NDA) and o-phthaldehyde (OPD), do not require GST for adduct formation with GSH. To examine the specificity of the dyes for GSH, cultures grown less than 6 DIV were pretreated with diethyl maleate or DL-buthionine-(S,R)-sulfoximine to deplete endogenous GSH. This resulted in a substantial loss of staining by CMAC, CMAC-blue, and MCB and partial loss of staining by OPD, indicating specificity for GSH, while staining by CMFDA or NDA was not altered, indicating a lack of specificity for GSH. Neurons experienced a dramatic decline in GSH levels relative to astrocytes between 5-6 DIV, as shown by a loss of neuronal staining with CMAC, CMAC-blue and MCB. This decrease in staining was not due to a decrease in GST activity, as neurons stained with the GST-insensitive OPD also exhibited a decline in GSH-sensitive staining. Immunolabeling experiments demonstrated that CMAC staining co-localized with GFAP-positive astrocytes, but not with MAP-2-positive neurons, in 18 DIV cultures. Finally, CMAC was exploited as a specific morphological marker of astrocytes in cultures aged >5 DIV. CMAC staining was employed to monitor astrocyte proliferation and to resolve astrocytes in living mixed cultures co-loaded with the Ca2+-sensitive dye, calcium green 5N-AM.

Original languageEnglish (US)
Pages (from-to)329-341
Number of pages13
JournalGlia
Volume30
Issue number4
DOIs
StatePublished - 2000

Keywords

  • Calcium
  • Cortical culture
  • Fluorophores
  • Glutathione-S-transferase
  • Morphology

ASJC Scopus subject areas

  • Neurology
  • Cellular and Molecular Neuroscience

Fingerprint Dive into the research topics of 'Evaluation of glutathione-sensitive fluorescent dyes in cortical culture'. Together they form a unique fingerprint.

  • Cite this

    Tauskela, J. S., Hewitt, K., Kang, L. P., Comas, T., Gendron, T., Hakim, A., Hogan, M., Durkin, J., & Morley, P. (2000). Evaluation of glutathione-sensitive fluorescent dyes in cortical culture. Glia, 30(4), 329-341. https://doi.org/10.1002/(SICI)1098-1136(200006)30:4<329::AID-GLIA20>3.0.CO;2-R