MiPNet17.17 Amplex-Mouse-brain

Fasching M, Sumbalova Z, Gnaiger E (2013) O2k-Fluorometry: HRR and H2O2 production in mouse brain mitochondria. Mitochondr Physiol Network 17.17.

» Open Access - File:MiPNet17.17 O2k-Fluorometry.pdf; O2k-Fluorescence

OROBOROS (2013) Mitochondr Physiol Network

Abstract: High-resolution respirometry [1] is extended by MultiSensor techniques allowing the simultaneous measurement of oxygen consumption and one additional parameter (mitochondrial membrane potential, pH, Ca²⁺, or NO concentration [2-5]). Fluorometric methods are available for a wide range of analytical parameters of major interest in mitochochondrial physiology: H₂O₂ production, mitochondrial membrane potential, intracellular pH, Ca²⁺, Mg²⁺, and NADH levels. Advantages of the simultaneous measurement of additional parameters in a single respirometric chamber:

1. Quality control of preparations by respirometric performance.
2. Objective exclusion criteria for protocol related problems, e.g. overtitration of uncouplers, side effects of TPP⁺ or fluorophores.
3. Elimination of artifacts of normalization for a mitochondrial marker when combining two methods in a single chamber.
4. Additional information is acquired for a limited amount of sample.
5. Oxygen dependence of parameters can be studied.

Here we introduce the O2k-Fluorescence LED2-Module, describe one selected stage in its development, and show its application for the simultaneous measurement of respiration and H₂O₂ production of mouse brain mitochondria.

• O2k-Network Lab: AT_Innsbruck_OROBOROS, AT Innsbruck Gnaiger E, AT Innsbruck MitoCom

Labels: MiParea: Respiration, Instruments;methods 

Organism: Mouse  Tissue;cell: Nervous system  Preparation: Isolated Mitochondria"Isolated Mitochondria" is not in the list (Intact organism, Intact organ, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP, Chloroplasts, Enzyme, Oxidase;biochemical oxidation, ...) of allowed values for the "Preparation" property. 

Regulation: Uncoupler  Coupling state: LEAK, OXPHOS, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property. 

HRR: Oxygraph-2k, Fluorometry"Fluorometry" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property., Protocol"Protocol" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property. 

References

1. Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopisies of human muscle. Methods Mol Biol 810: 25-58.
2. Aguirre E, Rodríguez-Juárez F, Bellelli A, Gnaiger E, Cadenas S (2010) Kinetic model of the inhibition of respiration by endogenous nitric oxide in intact cells. Biochim Biophys Acta 1797: 557-565.
3. MiPNetAnalyzer
4. Hickey AJ, Renshaw GM, Speers-Roesch B, Richards JG, Wang Y, Farrell AP, Brauner CJ (2012) A radical approach to beating hypoxia: depressed free radical release from heart fibres of the hypoxia-tolerant epaulette shark (Hemiscyllum ocellatum). J Comp Physiol B 182: 91-100.
5. Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2013) Combined high-resolution respirometry and fluorometry. Validation of safranin for determination of mitochondrial membrane potential. Methods Enzymol (in press).
6. Gnaiger E (2012) Mitochondrial Pathways and Respiratory Control. An Introduction to OXPHOS Analysis. 3rd ed. Mitochondr Physiol Network 17.18. OROBOROS MiPNet Publications, Innsbruck: 64 pp.