Question

A scientist carry out an experiment to examine living human fibroblast cells in cell culture and tag them with a membrane potential sensitive dye, TMRM. He completed measurements at 23 ºC using a confocal microscope. Observations yielded an average increase in dye brightness of 236-fold upon adding oligomycin to the mitochondria in the cells. The initial internal concentration of the TMRM dye was 9,271 nM. The external (extracellular) dye concentration remained unchanged at 154 nM. What is the membrane potential of the inhibited, but fully energized mitochondria? F = faraday constant = 96,487 coulombs mol-1 = 96.487 kJ mol-1 V-1 = 23.06 kcal mol-1 V-1; R (gas constant) = 8. 3143 J deg-1 mol-1; (delta psi) = - (RT/F) ln (Cin/ Cout ).

Answer 1

Currently I harvest cultured cells using trypsin, ressuspend cells in isotonic sucrose buffer (Sucrose 0.25M, EDTA 1mM, HEPES 10mM pH 7.4 with freshly added protease inhibitor cocktail), lyse cells using a Dounce homogenizer and use the centrifugation method in order to isolate my mitochondrial fraction.

Once I have my isolated fraction I ressuspend it in the same sucrose buffer previously described and am trying to determine if the isolated mitochondria has intact membrane or not.

I have been trying to use the "Isolated Mitochondria Staining Kit" from Sigma Aldrich that uses JC-1. From my understanding JC-1 monomers have green fluorescence and once accumulated into the mitochondria (due to it's transmembrane potential) it would form aggregates that shift to red fluorescence.

I was comparing results of mitochondrial fractions with and without treatment using valinomycin (which should dissipate mitochondria membrane potential) and obtained the same red fluorescence value for both.

I have also measured the green fluorescence of 1) Water 2) JC-1 solution 3) JC-1 solution + mitochondria fraction and 4) JC-1 solution + mitochondria fraction treated with valinomycin. Interestingly I got higher green fluorescence values for 3 and 4 and got much lower value for JC-1 solution, which didn't differ at all from water.