Evaluation of glutathione-sensitive fluorescent dyes in cortical culture.
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 Ca(2+)-sensitive dye, calcium green 5N-AM. GLIA 30:329-341, 2000. Published 2000 Wiley-Liss, Inc.
Tauskela
J S
JS
National Research Council of Canada, Institute for Biological Sciences, Ottawa, Ontario, Canada. joe.tauskela@nrc.ca
Hewitt
K
K
Kang
L P
LP
Comas
T
T
Gendron
T
T
Hakim
A
A
Hogan
M
M
Durkin
J
J
Morley
P
P
D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology* / Q000378:metabolism*; D005069:Evaluation Studies as Topic; D005452:Fluoresceins / Q000032:analysis* / Q000737:chemistry; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D051381:Rats; D012680:Sensitivity and Specificity
D005452:Fluoresceins; D005456:Fluorescent Dyes; D005904:Glial Fibrillary Acidic Protein; D005978:Glutathione