The overall goal of this procedure is to provide a simple method for simultaneously measuring mitochondrial calcium uptake and mitochondrial membrane potential in live cells using confocal microscopy. This method can help to answer key questions in the mitochondrial field, such as how mitochondria act as local calcium stores to tightly regulate intracellular calcium concentration. The main advantage of this technique is that both mitochondrial calcium and mitochondrial membrane potential can be specifically measured simultaneously in life cells without the need for genetic manipulation.
Though this method can provide insight into calcium regulation in mitochondrial disease pathology, it can also be used to study other newer degenerative conditions, when urinal calcium regulation in crucial. In this procedure, grow the cells on ten centimeter cell culture dishes, or 75 square centimeter flasks, in culture media at 37 degrees Celsius for 24 hours. To harvest the cells, remove media by aspiration, and wash the cells with five milliliters of PBS.
Afterward, remove PBS by aspiration. Then add 1.5 milliliters of Trypsin EDTA, and incubate the cells at 37 degrees Celsius for 2 minutes. Next tap the dish gently to remove the cells.
Resuspend them in five milliliters of culture media. Afterward, count the cells with a hemocytometer. The day before confocal imaging, plate the cells at 60 to 70 percent confluency in dishes or chambered cover slips.
Then incubate the cells overnight at 37 degrees Celsius to allow the cells to attach and recover. In this procedure, prepare the record staining solution with TMRM, Fluo 4 AM, and surfactant. Remove the culture media and wash the cells with 100 microliters of PBS.
Next remove PBS and incubate the cells in 100 microliters of record staining solution for 45 minutes at room temperature. Following that, remove the record staining solution and wash the cells in 100 microliters of calcium-free HBSS to remove excess Fluo 4 AM and TMRM. Then prepare the intracellular imagining solution with digitonin, TMRM, and thapsigargin.
Next remove the calcium-free HPSS and add 300 microliters of IM imaging solution to the cells. Leave the cells to equilibrate at room temperature for ten minutes before imaging with the calcium chloride additions. Use a low laser power of approximately 5 percent to minimize any photo damage to the cells.
Use the setting on the microscope lasers for the excitation and emission spectra of TMRM and Fluo 4 AM.Use a low laser power of approximately five percent with high gain to minimize photo damage of the cells. Next set the microscope to scan images every twenty five seconds. Scan the cells for ten minutes to establish the base line readings for TMRM and Fluo 4 signals.
Then pause the image scanning and add 3 microliters of 40 millimolar calcium chloride stock solution directly to the cells in 300 microliters of IM imaging solution. Mix gently with a pipette. When adding calcium solution to the cells, be careful not to move the dish on the microscope stage.
Continue image scanning for approximately four minutes. Then repeat calcium chloride additions every four minutes until the desired TMRM or Fluo 4 signals are reached, usually around eight to ten additions. Subsequently add a 1 to 100 dilution of one millimolar FCCP directly to the cells to dissipate the inner membrane potential.
Then image the cells for a further five minutes before the experiment is finished. Once the imaging is complete, determine the intensity of the Fluo 4 and TMRM signals using the image analysis software. To do so, open the image, click on the selection tool and select a region of interest that contains mitochondria.
Multiple regions of interest can be selected for measurement by opening Analyze, Tools, ROI Manager. Click Add to include the selected ROI for intensity measurement. Repeat previous steps until all ROIs have been added to the ROI manager.
Measure the average fluorescent intensity of the TMRM and Fluo 4 signals for each region of interest by selecting the more multi measure function. Make sure that the Measure All Slices is selected, and that One Row Per Slice is deselected. Then click Okay to obtain a table of mean fluorescent intensity of the TMRM and Fluo 4 signals for each ROI at each time point.
Then combine the data to calculate the average intensity and standard deviation for the TMRM and Fluo 4 signals. In this experiment, 143B cells were preloaded with Fluo 4 AM and TMRM before permeabilization with digitonin. Following each calcium chloride addition, the TMRM signal decreases as the influx of calcium ions into the mitochondria dissipates the mitochondrial membrane potential.
A small membrane potential is evident following permeability transition as the addition of 10 micromolar FCCP still induces a collapse of membrane potential. Here the mitochondrial calcium concentration, indicated by Fluo 4 signal, begins to increase following the second calcium chloride addition when the free calcium in the media reaches 0.32 micromolar. Each subsequent calcium chloride addition caused a progressive increase in mitochondrial calcium.
Shown here are the images of simultaneous measurements of membrane potential in mitochondrial calcium. The red signal indicates TMRM and the green signal indicates Fluo 4. Once mastered, this technique can be done in approximately two hours if performed properly.
After watching this video, you should have a good understanding of how to measure mitochondrial calcium levels and mitochondrial membrane potential simultaneously on confocal microscopy using the addition of exogenous calcium to the permeabilized cells. Don't forget that working with FCCP and digitonin can be extremely hazardous, and precautions such as wearing PPE should always been taking when performing this procedure.
