Talk:Safranin

Different methods for measurements of mt-membrane potential

Mt-membrane potential can either be measured with the O2k-Fluo LED2-Module by using the fluorophores TMRM or Safranin or with the O2k-TPP+ ISE-Module electrode by using the ion reporter TPP+. All mentioned ion reporters inhibit respiration, which makes it essential to test the optimum concentration before starting your experimenal series. Link zu QCS, terminology?

O2k-Fluo LED2-Module / Safranin and TMRM

This method is easy in handling since the Fluorescence-Sensor does not get in direct contact with the sample. The sensitivity of the fluorometric measurement is < 100 mV.

(Transformation of the fluorescence signal to mV need a method to be developed 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

O2k-TPP+ ISE-Module / TPP+ and TPMP

The O2k-TPP+ ISE-Module is a potentiometric method for determination of the mt-membrane potential by using the ion reporter TPP+ or TPMP and is more complex in handling than the O2k-Fluo Module. This method is suitable for measurements with a sensitivity > 100 mV MiPNet15.08_TPP-Electrode#Sensitivity_analysis_of_the_method and the ## can be determined directly by ###. Using the TPP-ISE electrode requires special care after the experiment, since the electrodes are inserted in the chamber and therefore gets in direct contact with the medium, substrates, uncouplers and also inhibitors. Standardized operating procedures help to avoid any carry-over.

(TPP+ shows the lowest inhibition from these dyes and can be used for the measurement of respiration with and membrane potential with CI substrates and CI&II substrates . The sensitivity of the TPP+ electrode could be low with CII substrate . TMRM or safranin could can be used safely only with a CII substrate.)

(From the signal of the TPP+ electrode, membrane potential in mV could can be calculated. Transformation of the fluorescence signal to mV need a method to be developed for each preparation and protein content, which has to be kept constant in all samples using this method .)