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 μM), by the trypan blue exclusion method (3±3 μM) or by MTT histochemistry (1±0.4 μM). Quantification of preOL viability with AB was unaffected by the presence of free radical scavengers (α-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.