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

The collection of blood samples for data and methods development presented herein was approved by the Internal Review Board of the University of Washington. Representative results also include data from male C57BL/6J mice (5-7 months old) purchased from Jackson Laboratories. All animal procedures were approved by the University of Washington Office of Animal Welfare. The protocol overview is pictured in Figure 1. Reagent preparation for this protocol can be found in Supplementary File 1.

Figure 1: Overview of the protocol. Workflow using high-resolution fluorespirometry to assess changes in mitochondrial membrane potential in isolated monocytes (CD14⁺) and T-cells (CD3⁺) from fresh human blood samples. Abbreviations: TMRM, tetramethylrhodamine methyl ester; SUIT, substrate-uncoupler-inhibitor titrations; ADP, adenosine diphosphate; Dig, digitonin; Mal, malate; Pyr, pyruvate; Glut, glutamate; D1-10, 10 consecutive ADP titrations. Please click here to view a larger version of this figure.

1. Separation of buffy coat from whole blood

NOTE: Cell isolation is modified from Kramer et al.²⁷.

1. Allow RPMI, density gradient, and centrifuge to reach room temperature. Sterilize the biosafety cabinet and materials before starting.
2. Collect venous blood into three 10 mL K₂EDTA tubes. Invert the tubes at least 3 times.
3. Centrifuge the tubes at 500 x g 10 min (22 °C, 9 acceleration [acc], 2 deceleration [dec]).
4. Remove 1 mL of plasma from each tube and store at -80 °C for future analyses.
5. Transfer the plasma and half the red blood cell layer from each tube into a single 50-mL conical tube. Add RPMI up to the 40 mL mark. Invert at least 3 times.
6. Slowly layer 10 mL of the plasma solution into four 15 mL conical tubes containing 3 mL of the density gradient.
7. Centrifuge at 700 x g for 30 min (22 °C, 5 acc, 2 dec).
8. Collect all the plasma and the buffy coat containing the peripheral mononuclear cells (PBMCs) without disrupting the red blood cells.
9. Centrifuge at 500 x g for 10 min (22 °C, 5 acc, 5 dec) and aspirate the supernatant.
10. Wash the PBMC pellet 1x-2x by resuspending it in 10 mL of RPMI and centrifuging at 500 x g for 10 min (22 °C, 5 acc, 5 dec).

2. Magnetic separation of CD14+ and CD3+ cells

1. Place a column in the magnetic field of a magnetic cell separator (see the Table of Materials). Wash the column with 3 mL of RP-5.
2. Resuspend the PBMC pellet in 80 µL of RP-5 and 20 µL of anti-CD14 microbeads (see the Table of Materials). Incubate for 15 min at 4 °C.
3. Resuspend the cells with 1 mL of RP-5 and load the suspension onto the column. Collect unlabeled cells that flow through into a 15 mL conical tube labeled "Flow-through 1". Wait until all cell suspension has gone through the column, then continue washing with 3 mL of RP-5 3x, collecting all flow-through.
4. Carefully remove the column from the magnetic field and place it on a new 15 mL conical tube. Add 5 mL of RP-5 and immediately use the plunger to purge the column contents into a collection tube labeled "CD14+".
5. Centrifuge "Flow-through 1" at 500 x g for 10 min (22 °C, 5 acc, 5 dec) and aspirate the supernatant.
6. Using cells from Flow-through 1, repeat steps 2.2-2.5 using anti-CD3 microbeads (see the Table of Materials) to isolate T-cells.
7. Centrifuge tubes containing T-cells (CD3⁺) and monocytes (CD14⁺) at 300 x g for 5 min. Aspirate the supernatant and resuspend the pellet in 1 mL of RP-5.
8. Determine cell concentration using a hemocytometer or automatic cell counter.
   NOTE: Cells can be counted by adding 10 µL of a cell dilution of 1:10 or 1:20 to a hemocytometer. One may refer to previously published protocols to count cells using a hemocytometer³⁶.
9. Pipette 2.5 million monocytes or 5 million T-cells into a new centrifuge tube. Centrifuge for 30 s at 2000 x g, aspirate the supernatant and resuspend the cells in MiR05 for a total volume of 20 µL and final concentration of 125 million monocytes or 250 million T-cells per mL.
   NOTE: The final concentration was selected to inject 2.5 million monocytes or 5 million T-cells in a volume of 20 µL. Mouse T-cells isolated from the spleen have also been tested using this method. The procedure is found in Supplementary File 1.

3. High-resolution fluorespirometry – Oxygen and TMRM fluorescence calibration

NOTE: This method was adapted from previous work done on permeabilized fibers by Pharaoh et al.¹¹. A high, un-inhibitory concentration of TMRM is used for quench mode, where the relationship of mMP and TMRM concentration in the matrix is inverted. Thus, a decrease in mMP leads to the release of TMRM dye from the matrix and an increase in fluorescence³².

1. Install 0.5 mL chambers in the O2K respirometer according to the manufacturer's instructions (see Table of Materials). Turn on the instrument and connect it to the software provided by the manufacturer for data acquisition.
2. Adjust the temperature to 37 °C and stir speed to 750 rpm.
3. Wash the chambers with distilled water 3x. Replace water with 0.54 mL of Mir05, close the stoppers fully, and remove excess buffer with the integrated suction system (ISS). Raise stoppers to allow room oxygen to equilibrate with chamber oxygen using a stopper-spacer.
4. Once the oxygen flux is stable, perform the air oxygen calibration (R1) according to the manufacturer's instructions.
   NOTE: It may take >30 min for oxygen flux to stabilize. Oxygen sensors require the determination of zero-point oxygen (R0) and background oxygen flux from 50-200 µM from separate experiments using dithionite titrations. Specific methods can be found in the manufacturer's manual.
5. Seal the chamber by closing the stoppers.
   NOTE: Unlike experiments using permeabilized fibers, the chambers do not require hyperoxygenation for PBMCs. Sealing the chamber after R1 calibration provides sufficient oxygen for the experiment. The oxygen levels should be maintained between 50-250 µM. If oxygen concentration falls below the threshold, the chamber can be partially opened so that chamber oxygen can equilibrate with room air oxygen.
6. TMRM calibration
   1. Use the Green LED fluo-sensors (ex. 525 nm) with the AmR filter set (see Table of Materials). Set fluorometer Gain için 1000 and Intensity için 1000. Turn on the fluo-sensors and begin recording the baseline.
   2. Inject 2.5 µL of 0.05 mM TMRM and allow the signal to stabilize (~2 min) before the next 2.5 µL injection until a total of 4 injections are performed for a total TMRM concentration of 1 µM TMRM in the chamber. Use a Hamilton syringe for all injections.
   3. Calibrate the fluo-sensor by selecting the fluorescent signal (voltage) for each injection representing 0, 0.25, 0.5, 0.75, and 1.0 µM of TMRM for a five-point calibration.

4. Substrate-uncoupler-inhibitor titration (SUIT) protocol

NOTE: Run blank experiments where 20 µL of Mir05 is injected into the chamber instead of 20 µL of cell suspension, as the TMRM signal will change in response to injections alone (discussed in representative results). Allow for the oxygen flux signal to stabilize (about 2-3 min) before the next injection for both blank and sample experiments. The following titration protocol and expected observations are in Table 1.

1. Once oxygen flux is stable, select and label both the oxygen flux and TMRM signal "pre-cell".
2. Inject the cell suspension containing either 5 million T-cells or 2.5 million monocytes in ~20 µL and measure for about 10 min. Select and label both the oxygen flux and TMRM signal as "Cell".
3. Permeabilize cells by injecting 2 µL of 1 mg/mL digitonin (final concentration: 4 µg/mL). Wait for 20 min. Select and label both the oxygen flux and TMRM signal as "Dig".
   NOTE: It is suggested to optimize the digitonin concentration in separate experiments.
4. Add 2.5 µL of 1 M succinate (final concentration: 5 mM). Select and label both the oxygen flux and TMRM signal as "SUCC".
5. Once oxygen flux is stable, add 5 µL of 100 mM malate (final concentration: 1.0 mM), 5 µL of 1 M glutamate (final concentration: 10 mM), and 5 µL of 500 mM pyruvate (final concentration: 5 mM). Select and label both the oxygen flux and TMRM signal as "MPG".
6. Once oxygen flux is stable, titrate ADP. Select rates for each titration and label them "D" sequentially 1 through 10, depending on the number of titrations. Use the titration scheme in Table 2.
7. Once oxygen flux is stable, perform a series of 1 µL titrations of 0.25 mM carbonyl cyanide p-(tri-fluromethoxy) phenyl-hydrazone (FCCP) until the fluorescent signal reaches its maximum. Select and label both the oxygen flux and TMRM signal representing minimum membrane potential and label it "FCCP".
   NOTE: FCCP concentration of 0.5-1.0 µM is usually required to deplete mitochondrial membrane potential.
   CAUTION: FCCP is toxic. Refer to the safety data sheet (SDS) for proper handling.
8. OPTIONAL: Once oxygen flux is stable, inject 1 µL of 0.25 mM rotenone to inhibit complex I and determine respiratory capacity through complex II.
   NOTE: Changes in membrane potential are no longer relevant after titrating with uncoupler.
   CAUTION: Rotenone is toxic. Refer to SDS for proper handling.
9. Once oxygen flux is stable, inject 1 µL of 1.25 mM (final concentration: 2.5 µM) of Antimycin A to inhibit mitochondrial respiration.
   CAUTION: Antimycin A is toxic. Refer to SDS for proper handling.

5. Calculation of mitochondrial membrane potential and analysis

1. Using blank experiments, record the calibrated TMRM values (micromolar TMRM) before the injection of the blank sample ("pre-sample") and for each of the injections. See Figure 2.
2. For each blank experiment, calculate the background ratio by setting the "pre-sample" TMRM concentration to 1.0. Calculate the subsequent proportional decrease in TMRM. Calculate the average background ratio from all blank experiments.
   NOTE: The number of blank experiments to include may depend on the instrument's precision. See the calculation example in Table 3 from five different blank experiments, where the standard deviation of the average background ratio fell between 0 and 0.016 for each titration.
3. Background calculation: Calculate the background for each sample experiment by multiplying "pre-sample" TMRM of the sample experiment times the average background ratio for each injection. See the calculation example in Table 3.
4. Background correction: Subtract the experiment's background to the measured TMRM values of the sample. See the calculation example in Table 4.
5. FCCP correction: Subtract the FCCP background-corrected mMP from each injection. See the calculation example in Table 4.
6. ADP sensitivity curve: Normalize the ADP-driven decrease in mMP by setting the highest and lowest membrane potential as 100% and 0%, respectively, using the mMP values collected throughout the ADP titration. Fit the data into a non-linear fit regression model using the preferred statistical software to calculate the half-maximal inhibitory concentration (IC₅₀) of ADP on mMP.
   NOTE: The curve fits the [Inhibitor] vs. normalized response – Variable slope in Prism.

Figure 2: Calculating mitochondrial membrane potential (mMP) and ADP sensitivity from TMRM fluorescence. Steps for calculating mitochondrial membrane potential (mMP) and ADP sensitivity from measurements of TMRM fluorescence by high-resolution fluorespirometry of one sample of T-cells (n = 1). Step 1: TMRM fluorescence is measured in blank samples as done in the biological sample. Step 2: Determine the ratio in the TMRM signal with each titration relative to the signal prior to the sample for each blank experiment. Calculate the average for each titration of all blank experiments. Step 3: Calculate the background for each sample experiment by multiplying the "pre-sample" fluorescence by the average background ratio for each titration. Step 4: Calculate the difference between background and sample TMRM fluorescence for each titration to express data as mMP or mitochondrial TMRM uptake. Step 5: Correct mMP so that full uncoupling with FCCP reflects zero mMP. Step 6: Perform non-linear regression to graph changes in mMP with increasing ADP concentrations. Measurements were performed in 0.5 mL chambers, one containing 5 million T-cells from a healthy volunteer. Averaged data are expressed as mean ± SEM. Single data points of a single replicate are expressed without error bars. Please click here to view a larger version of this figure.