A new Alamar Blue viability assay to rapidly quantify oligodendrocyte death.
We developed a rapid fluorometric viability assay for primary cultures of OL precursors (preOLs) or mature OLs that utilized the oxidation/reduction indicator dye Alamar Blue (AB). PreOLs had a lower rate of AB reduction than did mature OLs (0.02 +/- 0.01 units/min per cell versus 0.07 +/- 0.01). The assay was tested under two conditions toxic to preOLs: oxidative stress induced by glutathione depletion or kainate excitotoxicity. When glutathione was depleted by a 24-h exposure to cystine-depleted medium, the EC50 values for the dependence upon cystine for survival did not differ significantly when determined by AB reduction (2 +/- 2 microM), by the trypan blue exclusion method (3 +/- 3 microM) or by MTT histochemistry (1 +/- 0.4 microM). Quantification of preOL viability with AB was unaffected by the presence of free radical scavengers (alpha-tocopherol or idebenone) or the antioxidant enzymes Cu,Zn-superoxide dismutase and catalase. There was no difference in preOL viability as determined by AB or MTT after a 24-h exposure to kainate at concentrations up to 1 mM. AB offers a rapid objective measure of OL viability in primary culture and is a valid means to quantify OL death.
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Department of Neurology, Children's Hospital and Harvard Medical School, Boston, MA 02115, USA.
Khan
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R
Gan
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Rosenberg
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Volpe
J J
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D000818:Animals; D017209:Apoptosis / Q000502:physiology; D016923:Cell Death / Q000502:physiology*; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D004396:Coloring Agents; D009836:Oligodendroglia / Q000166:cytology*; D010078:Oxazines; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D014966:Xanthenes
D004396:Coloring Agents; D010078:Oxazines; D014966:Xanthenes; C005843:resazurin