Safranin

high-resolution terminology - matching measurements at high-resolution

Safranin

Description

Safranin is one of the most established dyes for measuring mitochondrial membrane potential by fluorometry. It is an extrinsic fluorophore with an excitation wavelength of 495 nm and emission wavelength of 587 nm. Safranin is a potent inhibitor of N-linked respiration and of the phosphorylation system.

Synonyms: Safranin O, Safranin Y, Saranin T, Gossypimine, Cotton Red, Basic Red2

Abbreviation: Saf

Reference: Kauppinen 1984 Am J Physiol, Krumschnabel 2014 Methods Enzymol, MiPNet20.13 Safranin mt-membranepotential

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Application in HRR

Safranin O (C₂₀H₁₉ClN₄)

Sigma S2255, 25 or 100 mg, store at -20°C; FW = 350.84

Caution: Light sensitive (store solution in a dark vial)!

Preparation of 1 mM stock solution

1. Weight 3.508 mg of Safranin and dissolve in distilled water to reach 10 mL final volume;
2. Divide into 0.5 mL portions (use dark vials);
3. Store at 4 °C.

At a concentration of 2 µM, safranin can be applied for simultaneous monitoring of oxygen consumption and mtMP of mt-preparations in a CII-linked substrate state.

O2k-Procedures: O2k-Fluorometry: HRR and simultaneous determination of mt-membrane potential with safranin. »MiPNet20.13«

Signal and output

1. Signal: The O2k-Fluo LED2-Module is operated through the amperometric (Amp)-Channel of the O2k, with electric current (ampere [A]) as the primary signal.
2. Output: The focus of the output with Safranin is on Type C: Force, mt-membrane potential.

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)
• CCCP (U)
• FCCP (U)
• Malonic acid (Mna)
• Antimycin A (Ama)
• DMSO
• Ethanol (EtOH)
• H₂O₂
• H₂O

HRR / Safranin and TMRM

The method is easy to use, since the Fluorescence-Sensor is not 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 an experimental series .

» More details: »Discussion«.

References

MitoPedia methods: Fluorometry