Probes
Tetramethyrhodamine ethyl ester (TMRE)
Rhodamine 123 (R123)
Rhodamine 6G (R6G)
These probes are membrane-permeable cationic probes, which are highly accumulated in mitochondria driving by mitochondrial membrane potential.
Since the highly accumulation, the concentration of these probes are quite high which is quite possible beyond its linear range and undergoes self-quenching. 5 uM was reported as a referral value of self-quenching for R123, similar for TMRE. Cellular toxicity of these probes was reported in previous studies. Based on previous publications, the toxicity of TMRE is less than R123, and Rhodamine 6 was the worst among these 3 probes.
These probes are also reported as subtrates of multi-drug efflux pump.
Rhodamine 123 (R123)
Rhodamine 6G (R6G)
These probes are membrane-permeable cationic probes, which are highly accumulated in mitochondria driving by mitochondrial membrane potential.
Since the highly accumulation, the concentration of these probes are quite high which is quite possible beyond its linear range and undergoes self-quenching. 5 uM was reported as a referral value of self-quenching for R123, similar for TMRE. Cellular toxicity of these probes was reported in previous studies. Based on previous publications, the toxicity of TMRE is less than R123, and Rhodamine 6 was the worst among these 3 probes.
These probes are also reported as subtrates of multi-drug efflux pump.
Cells
Bovine pulmonary arterial endothelial cells
General Experimental Protocol
Cell-coated beads was washed and suspended in 10mm x 10mm x 48mm acrylic fluorometric cuvettes containing HBSS/HEPES and R123 (10, 30, 100 or 500 nM) or TMRE (10, 20, 30, 100 and 500 nM).
The beads were allowed to settle to the bottom of the cuvette, and the dye concentration ([Re]) in the medium above the settled cells was measured (R123, lex = 490 nm, lem = 525 nm; TMRE, lex = 530 nm, lex = 573 nM) in 0, 10, 30, 60, 90, 120, 150 minutes for R123 and 0, 5, 10, 15, 20, 30, 40, 50 and 60 minutes for TMRE respectively.
The same protocol was carried out in the presence of the protonophore CCCP (5 mM), high K+ (138 mM KCl/5 mM NaCl) and/or the Pgp inhibitor GF120918 (2.5 mM) to check the effect of mitochondrial membrane potential, plasma membrane potential and efflux pump on probe distribution.
The same protocol was also carried out in the absence of cells to determine the contribution of nonspecific dye interactions with the plasticware.
The beads were allowed to settle to the bottom of the cuvette, and the dye concentration ([Re]) in the medium above the settled cells was measured (R123, lex = 490 nm, lem = 525 nm; TMRE, lex = 530 nm, lex = 573 nM) in 0, 10, 30, 60, 90, 120, 150 minutes for R123 and 0, 5, 10, 15, 20, 30, 40, 50 and 60 minutes for TMRE respectively.
The same protocol was carried out in the presence of the protonophore CCCP (5 mM), high K+ (138 mM KCl/5 mM NaCl) and/or the Pgp inhibitor GF120918 (2.5 mM) to check the effect of mitochondrial membrane potential, plasma membrane potential and efflux pump on probe distribution.
The same protocol was also carried out in the absence of cells to determine the contribution of nonspecific dye interactions with the plasticware.
Additional Measurements
% total cell LDH released into the medium were measured at the end of the experiments to assess cellular viability.
Cell bead weights were obtained by drying and weighing the beads at the end of each experiment to evaluate the cell surface area.
Cell bead weights were obtained by drying and weighing the beads at the end of each experiment to evaluate the cell surface area.
Results
Figure 1. The effect of cells on TMRE concentration in extracellular medium.
The initial concentration of TMRE in extracellular was 20 nM. The volume of microbeads covered by cultured bovine pulmonary arterial endothelial cells was round 0.17 mL. The concentration of TMRE was measured at 0, 5, 10, 15, 20, 30, 40, 50, 60 minutes respectively. |
Figure 2. The effect of initial TMRE concentration on the temporal change of TMRE in extracellular medium. Microbeads covered by cells were added into the medium with 10, 30 and 100 nM TMRE, the extracellular TMRE concentrations were measured and normalized to its initial concentration.
The experimental results show that the temporal course of TMRE redistribution was independent on its initial concentrations. |
Figure 3. The effect of inhibitors on TMRE redistribution in the presence of cells.
Portonpore CCCP (5 uM), efflux pump inhibitor GF120918 (2.5 uM) and high potasium (138 mM) were added to depress mitochondrial membrane potential, efflux pump and plasma membrane potential respectively. The results show that above factors play role in the redistribution of TMRE in the presence of cells. |
Figure 4. The effect of combined inhibitors on TMRE redistribution in the presence of cells.
Multiple inhibitors were added into the extracellular medium in order to break the high correlations between the factors affecting TMRE redistribution for following estimation using mathematical models. |
Figure 5. The minimum data set for the estimation of mitochondrial membrane potential
The set of experimental conditions: cells only, cell with GF120918, cells with GF120918 and high K+, cells with GF120918, high K+ and CCCP can break the correlations between mitochondrial membrane potential, plasma membrane potential and the capacity of multi-drug efflux pump on TMRE redistribution and contains sufficient information to estimate mitochondrial membrane potential, plasma membrane potential and the capacity of multi-drug efflux pump simultaneously. mitochondrial membrane potential: drives TMRE from cytosol to mitochodria Plasma membrane potential: drives TMRE from extracellular to cytosol Efflux pump: pumps TMRE from cytosol to extracellular medium. |