High-Resolution Fluorespirometry to Assess Dynamic Changes in Mitochondrial Membrane Potential in Human Immune Cells

Methods for studying mitochondrial bioenergetics under physiologically relevant substrate concentrations in immune cells are limited. We provide a detailed protocol that uses high-resolution fluorespirometry to assess changes in the response of the mitochondrial membrane potential to energy demand in human T-cells, monocytes, and peripheral mononuclear cells.

For a cell to function and survive following a period of physiological stress, it needs to be able to meet the energy demand required to restore homeostasis. In our lab, we seek to identify how mitochondria respond and adapt to nutritional stressors to better understand how mitochondria mediate the risk of developing disease. The current methods for testing PBMC bioenergetics involve measuring respiratory capacity after adding inhibitors and uncouplers.

These methods have helped us understand significant bioenergetic changes in PMBCs in different disease states. Membrane potential is essential for ATP synthesis and regulates processes such as respiratory flux, reactive oxygen species, and autophagy. However, we still have limited knowledge about how mitochondrial respiration and membrane potential together respond to physiological substrate concentrations in PBMCs.

The advantage of this technique is that it allows for an integrated analysis of mitochondrial membrane potential and oxygen consumption in human PBMC in response to increasing levels of ADP. This method allows for the quantification of the sensitivity of mitochondria to a shift in energy demand. To begin, install 0.5 milliliter chambers in the O2k respirometer according to the manufacturer's instructions.

Turn on the instrument and connect it to the provided software for data acquisition. Wash the chambers three times with deionized water. Replace the water with 0.54 milliliters of MIR O-5 and close the stoppers fully.

After removing the excess buffer, raise the stoppers to allow room oxygen to equilibrate with chamber oxygen. Place a column in the magnetic field of a magnetic cell separator, and wash the column with three milliliters of RP-5. Resuspend the freshly isolated peripheral blood mononuclear cell, or PBMC, pellet in 80 microliters of RP-5, along with 20 microliters of anti-CD14 microbeads.

Incubate the cells for 15 minutes at four degrees Celsius. Dilute the cells with one milliliter of RP-5 before loading the suspension onto the column. Collect unlabeled cells that flow through into a 15 milliliter conical tube, labeled as flow through one.

Wash the column three times with three milliliters of RP-5 to collect the flow through liquid. Carefully remove the column from the magnetic field and place it onto a new 15 milliliter conical tube. After adding five milliliters of RP-5, push the plunger into the column to collect contents into the tube.

After centrifuging the flow through one tube, repeat the isolation steps using anti-CD3 microbeads with the cells to obtain T lymphocytes. Centrifuge the cells containing CD3 positive T cells and CD14 positive monocytes. After discarding the supernatant, resuspend the pellet in one milliliter of RP-5.

Using a hemocytometer, determine the cell concentration. Add 2.5 million monocytes and 5 million T cells into new centrifuge tubes. Pellet the cells, and resuspend them in 20 microliters of MIR O-5.

Install the fluorescent sensors, and start an experimental file on the O2k respirometer unit. Using the green LED fluorescent sensors with the AMR filter set, adjust the fluorometer gain and the intensity to 1, 000. Set the chamber volume to 0.5 milliliters.

And adjust the layout to see both oxygen flux and TMRM fluorescence. Once the oxygen flux is stable, perform the air oxygen calibration according to the manufacturer's protocol. Close the stoppers to seal the chamber.

Using a Hamilton syringe, inject 2.5 microliters of 0.05 millimolar tetramethylrhodamine methyl ester, or TMRM, four times. Calibrate the flow sensor with a fluorescent signal for each injection representing zero, 0.25, 0.5, 0.75, and 1.0 micromolar of TMRM. Inject 20 microliters of MIR O-5 to run the blank experiment.

After the oxygen flux stabilizes, select and label both the oxygen flux and TMRM signal as pre-cell. Inject 20 microliters of the cell suspension containing the cells and measure for about 10 minutes. Then select and label both the oxygen flux and TMRM signals as cells.

Then repeat the steps for each treatment sequentially, and label the oxygen flux and TMRM signal appropriately. Once oxygen flux is stable, inject one microliter of 1.25 millimolar antimycin A to inhibit mitochondrial respiration. For each blank experiment, set the pre-sample TMRM concentration to one to calculate the background ratio.

Calculate the subsequent proportional decrease in TMRM and the average background ratio from all blank experiments. Then multiply the pre-sample TMRM of the sample experiment with the average background ratio for each injection of each sample experiment. Subtract the experiment's background to the measured TMRM values of the sample.

Next, subtract the FCCP background corrected mitochondrial membrane potential from each injection. To normalize the ADP-driven decrease, set the highest and lowest membrane potential as 100%and 0%respectively. And fit the data into a non-linear fit regression model.

Comparative studies on ADP-driven changes in T cells and monocytes from healthy volunteers showed that monocytes displayed greater loss of mitochondrial membrane potential as compared to T cells. The half maximal inhibitory concentration of ADP on the mitochondrial membrane potential was lower for monocytes as compared to the T cells. A typical dose response increase in oxygen consumption rates with ADP was not detected in either cell type.