TY - JOUR
T1 - High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria
AU - Rogers, George W.
AU - Brand, Martin D.
AU - Petrosyan, Susanna
AU - Ashok, Deepthi
AU - Elorza, Alvaro A.
AU - Ferrick, David A.
AU - Murphy, Anne N.
PY - 2011
Y1 - 2011
N2 - Recently developed technologies have enabled multi-well measurement of O 2 consumption, facilitating the rate of mitochondrial research, particularly regarding the mechanism of action of drugs and proteins that modulate metabolism. Among these technologies, the Seahorse XF24 Analyzer was designed for use with intact cells attached in a monolayer to a multi-well tissue culture plate. In order to have a high throughput assay system in which both energy demand and substrate availability can be tightly controlled, we have developed a protocol to expand the application of the XF24 Analyzer to include isolated mitochondria. Acquisition of optimal rates requires assay conditions that are unexpectedly distinct from those of conventional polarography. The optimized conditions, derived from experiments with isolated mouse liver mitochondria, allow multi-well assessment of rates of respiration and proton production by mitochondria attached to the bottom of the XF assay plate, and require extremely small quantities of material (1-10 μg of mitochondrial protein per well). Sequential measurement of basal, State 3, State 4, and uncoupler-stimulated respiration can be made in each well through additions of reagents from the injection ports. We describe optimization and validation of this technique using isolated mouse liver and rat heart mitochondria, and apply the approach to discover that inclusion of phosphatase inhibitors in the preparation of the heart mitochondria results in a specific decrease in rates of Complex I-dependent respiration. We believe this new technique will be particularly useful for drug screening and for generating previously unobtainable respiratory data on small mitochondrial samples.
AB - Recently developed technologies have enabled multi-well measurement of O 2 consumption, facilitating the rate of mitochondrial research, particularly regarding the mechanism of action of drugs and proteins that modulate metabolism. Among these technologies, the Seahorse XF24 Analyzer was designed for use with intact cells attached in a monolayer to a multi-well tissue culture plate. In order to have a high throughput assay system in which both energy demand and substrate availability can be tightly controlled, we have developed a protocol to expand the application of the XF24 Analyzer to include isolated mitochondria. Acquisition of optimal rates requires assay conditions that are unexpectedly distinct from those of conventional polarography. The optimized conditions, derived from experiments with isolated mouse liver mitochondria, allow multi-well assessment of rates of respiration and proton production by mitochondria attached to the bottom of the XF assay plate, and require extremely small quantities of material (1-10 μg of mitochondrial protein per well). Sequential measurement of basal, State 3, State 4, and uncoupler-stimulated respiration can be made in each well through additions of reagents from the injection ports. We describe optimization and validation of this technique using isolated mouse liver and rat heart mitochondria, and apply the approach to discover that inclusion of phosphatase inhibitors in the preparation of the heart mitochondria results in a specific decrease in rates of Complex I-dependent respiration. We believe this new technique will be particularly useful for drug screening and for generating previously unobtainable respiratory data on small mitochondrial samples.
UR - http://www.scopus.com/inward/record.url?scp=79960712083&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0021746
DO - 10.1371/journal.pone.0021746
M3 - Article
C2 - 21799747
AN - SCOPUS:79960712083
SN - 1932-6203
VL - 6
JO - PLoS ONE
JF - PLoS ONE
IS - 7
M1 - e21746
ER -