A rapid and sensitive assay for measuring mitochondrial metabolic activity in isolated neural tissue.
In the present study, we used the oxidation-reduction sensitive dye Alamar Blue, a fluorometric/colorimetric indicator of metabolic activity, as a tool for examining mitochondrial function in rat spinal cord synaptosomes. At 15 min following incubation, Alamar Blue fluorescence levels were found to increase by 3-fold, and could be detected in samples containing as little as 25 microg of protein. Alamar Blue is non-toxic, making it possible to obtain measures of the metabolic rate and the maximal functional capacity of mitochondria in a single sample. The findings of this study demonstrate that Alamar Blue fluorescence levels increased in a near linear fashion when samples were measured every 15 min for a period of 1 h. To document that the changes in Alamar Blue fluorescence are directly related to mitochondrial function, synaptosomes were pre-incubated with antimycin A (10 microM) or malonate (50 mM), both of which are potent inhibitors of mitochondrial function. Pretreatment with either compound significantly reduced the Alamar Blue fluorometric signal at all time points examined. These results provide evidence that Alamar Blue is a valuable analytical tool for examining mitochondrial function in synaptosomal preparations from neural tissue. Moreover, the properties of Alamar Blue are such that it provides a more sensitive and simpler indicator compared to indicators used in existing assays.
Springer
J E
JE
Department of Anatomy and Neurobiology, University of Kentucky Medical Center, 800 Rose Street, Lexington, KY 40536-0084, USA. jspring@pop.uky.edu
Azbill
R D
RD
Carlson
S L
SL
D000818:Animals; D004396:Coloring Agents; D005260:Female; D005470:Fluorometry; D066298:In Vitro Techniques; D016014:Linear Models; D008928:Mitochondria / Q000378:metabolism*; D010078:Oxazines; D010084:Oxidation-Reduction; D051381:Rats; D012680:Sensitivity and Specificity; D013116:Spinal Cord / Q000378:metabolism* / Q000648:ultrastructure; D013574:Synaptosomes / Q000378:metabolism*; D013997:Time Factors; D014966:Xanthenes
D004396:Coloring Agents; D010078:Oxazines; D014966:Xanthenes; C005843:resazurin