Materials and Reagents

15 ml, 50 ml screw cap tubes (SARSTEDT, catalog numbers: 62.554.502 ; 62.547.254 )
1.5 ml, 2 ml reaction tubes (SARSTEDT, catalog numbers: 72.706 ; 72.695.500 )
20 µl, 200 µl, 1,000 µl pipette tips (SARSTEDT, catalog numbers: 70.1116 ; 70.760.002 ; 70.762 )
5 ml, 10 ml, 25 ml serological pipettes (SARSTEDT, catalog numbers: 86.1253.001 ; 86.1254.001 ; 86.1685.001 )
50 ml Leucosep^TM tubes (Greiner Bio-One GmbH, catalog number: 227290 )
Seahorse XF24 FluxPak containing XF24 sensor cartridges, XF24 cell culture microplates and XF Calibrant Solution (Agilent Technologies, catalog number: 100850-001 )
Human venous blood, EDTA-anticoagulated – S-Monovette^® 7.5 ml K3E (SARSTEDT, catalog number: 01.1605.001 )
Ficoll-Paque^TM PLUS (GE Healthcare, catalog number: 17-1440-03 )
Poly-D-Lysine solution, 1.0 mg/ml (Merck Millipore, catalog number: A-003-E , storage temp. -20 °C)
Trypan Blue solution 0.4% (Sigma-Aldrich, catalog number: 93595 )
Dulbecco’s Modified Eagle’s Medium (DMEM), high glucose (Sigma-Aldrich, catalog number: D7777 , storage temp. 4 °C)
Dimethyl sulfoxide (DMSO) (Carl Roth, catalog number: A994 )
Oligomycin from Streptomyces diastatochromogenes (Sigma-Aldrich, catalog number: O4876 , storage temp. -20 °C; mixture of isomers A, B, and C), 2.5 mM in DMSO
Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; Sigma-Aldrich, catalog number: C2920 , storage temp. 4 °C), 2.5 mM in DMSO
Rotenone (Sigma-Aldrich, catalog number: R8875 ), 2.5 mM in DMSO
Antimycin A from Streptomyces sp. (Sigma-Aldrich, catalog number: A8674 , storage temp. -20 °C), 2.5 mM in DMSO
NaCl (Carl Roth, catalog number: 3957 )
NaOH (Carl Roth, catalog number: 6771 )
KCl (Carl Roth, catalog number: 6781 )
Na₂HPO₄·12H₂O (Carl Roth, catalog number: N350 )
KH₂PO₄ (Carl Roth, catalog number: 3904 )
1× PBS (see Recipes)
0.9% NaCl solution (see Recipes)
10 M NaOH solution (see Recipes)
Poly-D-Lysine working solution (50 µg/ml) (see Recipes)
2.5 mM oligomycin solution (see Recipes)
2.5 mM FCCP solution (see Recipes)
0.1 M rotenone solution (see Recipes)
2.5 mM rotenone solution (see Recipes)
2.5 mM antimycin A solution (see Recipes)
Assay medium (see Recipes)

Equipment

Pipettes: Eppendorf Research^® Plus 10 μl, 20 µl, 200 µl, 1,000 µl (Eppendorf, catalog numbers: 3123000020 ; 3123000039 ; 3123000055 ; 3123000063 )
Pipetting aid: PipetBoy acu 2 (Integra Biosciences, catalog number: 155 000)
Neubauer counting chamber improved (Carl Roth, catalog number: PC72.1 )
Inverted microscope: Primovert (Carl Zeiss, catalog number: 415510-1100-000 )
Water bath (GFL, catalog number: 1003 )
Incubator without CO₂ (GFL, catalog number: 4010 )
Swing out rotor centrifuge: 5804 R (Eppendorf, catalog numbers: 5805000010 ; 5804709004 )
Water purification system: ELGA^® PURELAB flex 3 (Veolia, catalog number: PF3XXXXM1 )
pH meter: FE20 FiveEasy^TM (Mettler Toledo, cataog number: 30266626 )
Seahorse XF24 Extracellular Flux Analyzer (Agilent Technologies, catalog number: 100737-100 )

Software

Wave Controller Software (Agilent Technologies, version 1.8.1.1)
Optional: Wave Desktop Software (Agilent Technologies)

Procedure

The experimental procedure for the detection of mitochondrial function in human PBMCs using a XF Analyzer is shown schematically in Figure 2. This is followed by a detailed description of the individual work steps.

Figure 2. Workflow diagram of mitochondrial function measurement in human PBMCs using a XF Analyzer

Day before the experiment
1. Hydration of XF24 sensor cartridge (consisting of the cartridge lid, the sensor cartridge itself and the utility plate; see Figure 3B and 3C):
  1. Add 1 ml XF Calibrant Solution into each well of the utility plate.
  2. Remove air bubbles that might arise at the sensors with a pipette tip.
  3. Incubate the assembled plate in a non-CO₂ 37 °C incubator overnight.
2. Switch on the XF24 Analyzer (see Figure 3A) and let it warm up to 37 °C overnight.
  
  Figure 3. XF24 Analyzer (A) and XF24 sensor cartridge (B and C). The sensor cartridge, including markings of the sensors, is shown from above (B) as well as from below (C). In addition, the injection ports are marked (B) and the utility plate is depicted (C).
3. Programming of measurement protocol on the XF Analyzer:
  1. Equilibration: 30 min
  2. Measurement period 1:
    1. Mixing: 4 min
    2. Waiting: 2 min
    3. Measuring: 3 min
      → 3 repetitions
  3. Injection of port A (50 µl of 7.5 µM oligomycin; final concentration 0.75 µM)
  4. Measurement period 2:
    1. Mixing: 4 min
    2. Waiting: 2 min
    3. Measuring: 3 min
      → 3 repetitions
  5. Injection of port B (55 µl of 10 µM FCCP; final concentration 1 µM)
  6. Measurement period 3:
    1. Mixing: 4 min
    2. Waiting: 2 min
    3. Measuring: 3 min
      → 3 repetitions
  7. Injection of port C (60 µl of 16.7 µM rotenone/antimycin A; final concentration 1.67 µM)
  8. Measurement period 4:
    1. Mixing: 4 min
    2. Waiting: 2 min
    3. Measuring: 3 min
      → 3 repetitions

Day of the experiment
1. Coating of XF24 cell culture microplate:
  1. Add 30 µl of Poly-D-Lysine working solution (50 µg/ml) into each well and incubate the plate for 1 h at RT.
  2. Discard the supernatant, wash each well with 300 µl ddH₂O and let the plate air dry under sterile conditions (approximately 30 min).
2. Isolation of PBMCs (continue with this step during microplate coating; see Figure 4):
  1. Add 15 ml of Ficoll-Paque^TM PLUS (RT) into a 50 ml Leucosep^TM tube and centrifugate at 1,000 × g for 30 s at RT.
  2. Dilute an appropriate volume of anticoagulated human blood at a ratio of 1:2 with 0.9% NaCl solution (at least 7.5 ml and at most 15 ml).
  3. Transfer the resulting volume (at least 15 ml and at most 30 ml) into the prepared Leucosep^TM tube and centrifuge at 1,000 × g for 10 min at RT in a swing out rotor with brakes switched off.
  4. Harvest the PBMC fraction which appears as white layer between the plasma and the Ficoll above the porous barrier of the Leucosep^TM tube [for detailed information see manufacturer's instructions or refer to Matt and Bergemann, (2019)]. To do this use a 1,000 µl Eppendorf pipette and collect the PBMCs in a 50 ml screw cap tube.
  5. Wash the cells once with 10 ml PBS and centrifuge at 250 × g for 10 min.
  6. Repeat the washing step twice with 5 ml PBS each.
  7. Determine the number of cells using a Neubauer counting chamber improved.
  Figure 4. Isolation of PBMCs with a Leucosep^TM tube. On the left side, the tube is filled with Ficoll-Paque^TM below and anticoagulated and diluted blood above the porous barrier. On the right side, after density gradient centrifugation, the tube shows the following layers from top to bottom: plasma – PBMCs – Ficoll-Paque^TM – porous barrier – Ficoll-Paque^TM – erythrocytes and granulocytes.
3. Prepare the assay medium and preheat at 37 °C before use.
4. Seeding PBMCs onto pre-coated XF24 cell culture microplate:
  Note: 4 to 5 wells per condition are recommended. The wells A1, B4, C3 and D6 are normally used as background wells (without cells). An exemplary plate layout as well as an image of PBMCs seeded on Poly-D-Lysine is shown in Figure 5.
  1. Take 5 × 10⁵ of the isolated PBMCs per well and centrifuge at 250 × g for 5 min.
  2. Resuspend the cell pellet in 100 µl assay medium per well.
  3. Add 100 µl of cell suspension per well.
  4. Fill the background wells with 450 µl assay medium.
  5. Centrifuge the plate at 250 × g for 1 min in a swing out rotor with brakes switched off.
  6. Add 350 µl assay medium to the wells with cells (final volume in each well: 450 µl).
  7. Incubate the plate at 37 °C without CO₂until measurement (on average about 1 h).
    
    Figure 5. Exemplary plate layout of an XF24 cell culture microplate (left side) and a bright field microscopy image of PBMCs seeded on a Poly-D-Lysine coated XF24 cell culture microplate (right side). Left side: The wells A1, B4, C3 and D6 are used as background wells (without cells). One color (light green, dark green, dark orange, light orange) represents one condition each. There are 5 wells per condition. Right side: The image shows PBMCs in 100× magnification.
5. Loading of XF24 sensor cartridge with compounds:
  1. Prepare compounds as follows:
    1. Dilute 4.86 µl of 2.5 mM oligomycin solution in 1,615 µl assay medium (results in 7.5 µM).
    2. Dilute 6.48 µl of 2.5 mM FCCP solution in 1,614 µl assay medium (results in 10 µM).
    3. Dilute 10.82 µl of 2.5 mM rotenone solution and 10.82 µl of 2.5 mM antimycin A solution in 1,598 µl assay medium (results in 16.7 µM each).
  2. Load the injection ports of the XF24 sensor cartridge as follows:
    Note: Avoid the formation of air bubbles in the injection ports and handle the XF24 sensor cartridge with care after loading in order to prevent dripping down of the compounds.
    1. Fill all ports A with 50 µl of 7.5 µM oligomycin.
    2. Fill all ports B with 55 µl of 10 µM FCCP.
    3. Fill all ports C with 60 µl of 16.7 µM rotenone/antimycin A.
6. Start the run in the XF Analyzer (follow the instructions of the software):
  1. Place the XF24 sensor cartridge (without lid) in the XF Analyzer and start the calibration.
  2. After completion of the calibration, replace the utility plate by the XF24 cell culture microplate and start measurement.
7. Normalization of measurement results to the number of cells:
  1. Resuspend PBMCs in the final volume of each well (615 µl) by repeatedly pipetting up and down.
  2. Determine the number of cells per well using a Neubauer counting chamber improved.

Data analysis

Divide the counted number of cells per well by 10,000 and enter the results in the Wave software under ‘Normalize’ in order to normalize the data (see Figure 6).

Figure 6. Screenshot of the Wave software with markings for normalization and data export

Define outliers based on comparing the course of the OCR of single wells of one condition and exclude them from analysis.
Note: At least three replicates should be used for analysis.
Export the results to the Seahorse XF Cell Mito Stress Test Report Generator, which is linked to the Wave software (see Figure 6). This software tool automatically calculates and reports the parameters, which are analyzed by the Mito Stress Test.
Take the calculated values from the Report Generator and analyze them with an appropriate statistical software.

Representative Data
A typical result of an oxygen consumption rate measurement using a XF Analyzer and the stress reagents oligomycin, FCCP, antimycin A and rotenone (Mito Stress Test) is shown in Figure 7.

Figure 7. Oxygen consumption rate (OCR) of a Mito Stress Test of PBMCs measured with a XF Analyzer. The results of two different subjects are depicted not normalized (A) and normalized (B).

Notes

Since human blood samples are often available only once per condition, the measurement of a standard PBMC sample, which is stored deep-frozen, is recommended. This can be used for an additional normalization step.
Port D of the XF24 sensor cartridge is not used in the described experiment and therefore all ports D can be left blank. Of the ports A, B and C all 24 wells have to be filled in order to ensure an equal injection of the compounds.

Recipes

1× PBS
8 g NaCl
0.20 g KCl
2.88 g Na₂HPO₄·12H₂O
1.24 g KH₂PO₄
ddH₂O to 1 L, pH 7.4
0.9% NaCl solution
9 g NaCl
ddH₂O to 1 L
10 M NaOH solution
40 g NaOH
ddH₂O to 100 ml
Poly-D-Lysine working solution (50 µg/ml)
50 µl Poly-D-Lysine solution, 1.0 mg/ml
950 µl ddH₂O
2.5 mM oligomycin solution
5 mg oligomycin
2.528 ml DMSO
2.5 mM FCCP solution
10 mg FCCP
15.737 ml DMSO
0.1 M rotenone solution
1 g rotenone
25.353 ml DMSO
2.5 mM rotenone solution
25 µl of 0.1 M rotenone solution
975 µl DMSO
2.5 mM antimycin A solution
25 mg antimycin A
18.529 ml DMSO
Assay medium
0.675 g Dulbecco’s Modified Eagle’s Medium, high glucose
ddH₂O to 50 ml, pH 7.4

Acknowledgments

This work was funded by the Baden-Württemberg Ministry of Science, Research and Art via the “Cooperative Graduate School InViTe”. We like to thank Dr. med. Adrian Schulte and his team of the F. X. Mayr Bodensee Centre in Überlingen-Hödingen for providing the blood samples.

Competing interests

The authors declare no competing interests.

Ethics

The experiments were conducted in accordance with the declaration of Helsinki and approved by the ethics committee of the Landesärztekammer Baden-Württemberg, Germany. Furthermore, all volunteers were informed in advance and gave their written consent to the use of their blood samples.

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