UV excitable plural probes, have an inherent signal contamination, because of the auto-plural sounds. Most of the problem with NADH, is limiting the use of a plural probe. A method can be used, to precisely correct the outer florescence from NADH, in order to extract a true, uncontaminated plural probe signal.
Demonstrating the procedure will be Jeong Hoon Lee, our research assistant professor from my laboratory. After allowing the myocytes to settle to the bottom of the tube, aspirate the supernatant. Label the cells with two milliliters of freshly prepared one millimolar fura-2-FFAM diluting solution for 60 minutes at four degrees Celsius.
At the end of the incubation, place the tube in a 37 degree Celsius water bath for 30 minutes in the upright position before removing the supernatant. Then add four milliliters of room temperature culture medium for storage at room temperature. For in situ isobestic fura-2-ffa point identification mount the diluted cells onto the microscope stage and wait three minutes for the cells to sink to the bottom.
Profuse NADH-free 37 degree Celsius calcium-free solution into the cell culture and locate a target cell. Profuse saponin solution for 60 seconds before re-profusing with the NADH-free calcium-free solution. Simultaneously measure the fura-2-ff emitted signals at 450 and 500 nanometers, using an excitation scan from 350 to 365 nanometers with a 0.1 nanometer step and re-profuse with NADH-free calcium-free solution.
Next subtract the signals in the calcium saturated solution from the signals in the calcium free conditions. Then calculate the standard deviations of the emission at each excitation and select the excitation wavelength showing the minimum standard deviation values as individual isobestic points. To use a multi-parametric measurement system to detect the background signal and to correct the signal within the cell area, mount dye-free cells in the microscope bath and allow the cells to settle for three minutes.
Profuse NADH calcium-free solution into the bath for about five minutes at a two to three milliliters per minute rate and set the object field to cover the targeted cell. After moving the cell out of the field measure the background signals of the cell-free window and set the signals as offsets. Then return the cell to the initial position and measure the cell background signals and the cell area.
To calculate the R-factor use the acquired cell background and area signals before remounting the dye-free cells onto the microscope and profusing with calcium-free solution. Measure the signals as indicated before profusing the cell suspension with malate solution. After measuring the signals profuse the cells with pyruvate solution followed by profusion with malate-pyruvate solution and rotenone solution.
Then calculate the slope for each signal. Here the changes in mitochondrial calcium before and after the correction are shown. The results clearly reveal substantial changes in the mitochondrial calcium with a mitochondrial resting calcium concentration without cytosolic calcium of 1.03 plus or minus 0.13 micromolar and maximum mitochondrial calcium at one micromolar of calcium of 29.6 plus or minus 1.61 micromolar.
The mitochondrial transmembrane potential was monitored with a profusion of two nanomolars TMRE. The change in mitochondrial potential was calculated based on an assumed initial mitochondrial membrane potential of minus 150 millivolts, demonstrating a decrease in NADH in response to calcium addition, with a negliable effect on the mitochondrial membrane potential. The mitochondiral pH was not effected by the increase in mitochondrial calcium induced by carboxy SNARF-1 loading.
Incorrect isobestic points result in incorrect R and NADH correction. So take care to always correctly measure the cell-free background and cell area. This technique is used to prepared mitochondrial calcium dynamic studies in the bioanalytical spills.
