MiPNet20.13 Safranin mt-membranepotential

O2k-Fluorometry: HRR and simultaneous determination of mt-membrane potential with safranin.

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Oroboros (2015-06-15) Mitochondr Physiol Network

Abstract: Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2015) O2k-Fluorometry: HRR and simultaneous determination of mt-membrane potential with safranin. Mitochondr Physiol Network 20.13(01):1-5. »Versions

Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81. »Bioblast link«

• O2k-Network Lab: AT_Innsbruck_Oroboros

Labels: MiParea: Respiration, Instruments;methods

Organism: Mouse Tissue;cell: Nervous system Preparation: Homogenate

Coupling state: LEAK, ET Pathway: S, ROX HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Protocol

O2k-Demo, O2k-MultiSensor, MitoFitPublication

MitoPedia O2k and high-resolution respirometry: O2k-Open Support

» The Oroboros MitoLab uses Safranin O from Sigma (#S2255, 25 g).

» More details: »Discussion«.

Safranin chemical background

Several substances typically used in SUIT protocols may influence the fluorescence signal of safranin when injected into the O2k-Chamber (for instance coloured substances such as cytochrome c). These chemicals should be tested for their effect in a background run without biological sample, and the necessary corrections be applied.

Substances with an effect on the fluorescence signal of safranin

ADP (D)
Cytochrome c (c)
Succinate (S)
Rotenone (Rot)
Ascorbate (As)
TMPD (Tm)

Substances without an effect on the fluorescence signal of safranin

Pyruvate (P)
Malate (M)
Glutamate (G)
Digitonin (Dig)
Oligomycin (Omy)
FCCP (U)
Malonic acid (Mna)
Antimycin A (Ama)
DMSO
Ethanol
H₂O₂
H₂O

O2k-Fluo LED2-Module / Safranin and TMRM

The method is easy to use, since the Fluorescence-Sensor does not get in direct contact with the sample. Transformation of the fluorescence signal of safranin or TMRM to mtMP [mV] requires a specific method for each preparation and protein content, which has to be kept constant in all samples using this method.

References:

Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81. - »Bioblast link«
Sumbalova Z, Gnaiger E (2015) High-resolution measurement of mitochondrial membrane potential and respiration – comparison of potentiometric and fluorometric methods. Abstract MiP2015. »Bioblast link«