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
- 1 Description
- 2 Application in HRR
- 3 Signal and output
- 4 Safranin chemical background
- 5 Calibration of the raw fluorescence signal
- 6 Analysis of mitochondrial membrane potential using safranin
- 7 HRR / Safranin and TMRM
- 8 References
MitoPedia O2k and high-resolution respirometry: O2k-Open Support
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
- Weight 3.508 mg of Safranin and dissolve in distilled water to reach 10 mL final volume;
- Divide into 0.5 mL portions (use dark vials);
- Store at 4 °C.
- Preparation of 1 mM stock solution
- 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
- 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.
- Fluo-Sensor: Smart Fluo-Sensor Blue equipped with Filter Set Saf
- 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
Substances without an effect on the fluorescence signal of safranin
Calibration of the raw fluorescence signal
- To calibrate the raw fluorescence signal expressed as [V] and convert to µM, you need to click on ´Calibration/A: or B: Amperometric, Amp´ to open´[Amp calibration-DatLab| Amp calibration]´ window in DatLab.
Analysis of mitochondrial membrane potential using safranin
- Data analysis of mitochondrial membrane potential estimation using various fluorescence dyes: MiPNet24.09 General Template for Mt-membrane Potential Analysis to calculate the relative mt-membrane potential values.
- The calculation used to calculate the mt-membrane potential values in the Excel tempalte are provided here complying with Oroboros transparancy policy:
- cadded: titrated, free concentration of safranin [M=mmol/L]
- cext,free : free concentration of safranin outside mitochondria [M=mmol/L]
- ctotal: total concentration of safranin in the O2k-Chamber corrected for titration volume [M=mmol/L]
- ctotal,initial: total, initial concentration of safranin in the O2k-Chamber [M=mmol/L]
- Ki': apparent partition coefficient describing internal binding
- Ko': apparent partition coefficient describing external binding
- nadd : total amount of probe ions added to the system [µmol]
- Pmt: total mitochondrial protein content (as a marker for mitochondrial membrane content); in the case of isolated mitochondria: Pmt=Pc.
- PC: total cellular protein content (as a marker for cellular membrane and other material content); note that the mitochondrial protein content is accounted twice: in Pmt and in PC, this may be partially justified: while the outside of the mitochondrial membrane contributes to the external binding, the inside contributes to internal binding. However, also the outside of the cell membrane contributes twice. There is probably no numerical significance in this question, considering the low impact of external binding at reasonable high membrane potentials
- Vmt(abs): total mitochondrial volume in the system (O2k-Chamber) [µL]
- Vmt(spec): mass specific mitochondrial matrix volume (per mass of mitochondrial protein), [1µL/mg]
- Vext : external volume: total solution volume outside mitochondria [µL]
- Vinj : titration volume [µL]
- Vtotal: O2k-Chamber volume [µL]
- Fbound,int: internal binding ratio; ratio of internally bound probe molecule to total amount of probe molecules taken up by the mitochondria
- Fbound,ext: external binding ratio; ratio of externally bound probe molecule to total amount of probe molecules outside of mitochondria
Correction for chemical background in the `1-calibration and chemical BG´excel sheet
- 1. Diluted Amp signal: diluted free safranin concentration corrected for chemical titration volumes
- 2. Titration correction of the diluted Amp signal:
- Titration effect of chemicals on the Amp signal calculated from the diluted Amp signal
Amp signaldiluted2- Amp signaldiluted1
- Amp signaldiluted2: Amp signal after the chemical titration corrected for the titration volume
- Amp signaldiluted1: Amp signal before the chemical titration corrected for the titration volume
- 3. Titration correction of the measured Amp signal:
Titration effect of chemicals on the Amp signal calculated from the measured Amp signal
Amp signalmeasured2- Amp signalmeasured1
- Amp signalmeasured2: Amp signal after the chemical titration corrected for the titration volume
- Amp signalmeasured1: Amp signal before the chemical titration corrected for the titration volume Background correction factor= Titration correction of the measured Amp signal- Titration correction of the diluted Amp signal
- 4. Correction factor: correction factor for the chemical background
Titration correction of the measured Amp signal- Titration correction of the diluted Amp signal
Correction for chemical background in the `2-biol exp calculation´excel sheet
Cumulative BG correction factor [µM]= Cumulative BG correction factorbefore + background correction factorafter
- Cumulative BG correction factorbefore: calculated cumulative BG correction factor of the previous titration
- Background correction factorafter: background correction factor after the titration
Corrected Amp signal=Amp signal- cumulative BG correction factor = cout,free
- For more detailed explanation, see MiPNet24.11 mtMP calculation
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«.
- Bioblast links: Force and membrane potential - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
.::: Fundamental relationships
- 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«
MitoPedia methods: Fluorometry
Labels: MiParea: Instruments;methods
Regulation: mt-Membrane potential
HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Protocol, O2k-FluoRespirometer"O2k-FluoRespirometer" 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.
O2k-Core, DatLab, Safranin