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Jan 2020

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Ex vivo Assessment of Mitochondrial Function in Human Peripheral Blood Mononuclear Cells Using XF Analyzer
使用XF分析仪体外评估人外周血单个核细胞线粒体功能   

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Abstract

Cellular health and function, as we know today, depend on a large extent on mitochondrial function. The essential function of mitochondria is the energy production, more precisely ATP production, via oxidative phosphorylation. Mitochondrial energy production parameters therefore represent important biomarkers. Studies on human cells have mainly been performed on in vitro cell cultures. However, peripheral blood mononuclear cells (PBMCs) are particularly suitable for such examinations. That’s why this protocol describes a method to measure key parameters of mitochondrial function in freshly isolated PBMCs with the latest technology, the XF Analyzer. For this ex vivo approach PBMCs are first isolated out of human anticoagulated blood. Next, they are attached to the surface of special microplates pre-coated with Poly-D-Lysine. During the subsequent measurement of oxygen consumption rate (OCR) as well as extracellular acidification rate (ECAR) the stress reagents oligomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), rotenone and antimycin A are injected. Several mitochondrial parameters can be calculated from the results obtained. The application of this protocol allows the analysis of various influences, such as pharmaceuticals or environmental factors, on human cells.

Keywords: Human peripheral blood mononuclear cells (PBMCs) (人外周血单核细胞), Mitochondria (线粒体), XF Analyzer (XF 分析仪), XF Cell Mito Stress Test (XF线粒体压力测试), Oxygen consumption rate (耗氧率), Mitochondrial respiration (线粒体呼吸), Ex vivo (体外)

Background

Mitochondria play a critical role in maintaining normal cellular function. It is now common knowledge that they not only produce ATP via oxidative phosphorylation but, for example, are also involved in the metabolism of amino acids, lipids and nucleotides, diverse signaling and redox processes as well as quality control and degradation processes including mitophagy and apoptosis (Pfanner et al., 2019). However, mitochondria represent the major site of ATP synthesis in normal cells (Akbari et al., 2019). For this purpose, an electrochemical proton gradient is generated across the mitochondrial inner membrane through the multi-subunit enzyme complexes I–IV. This proton gradient is used by the ATP synthase, also known as complex V, to turn ADP into ATP (Chaban et al., 2014).


The process of oxidative phosphorylation is associated with the reduction of oxygen to water. Accordingly, the oxygen consumption rate of cells can be used for assessing mitochondrial function (Smolina et al., 2017). This principle is the basis of Seahorse XF Analyzers (Agilent Technologies). They provide the possibility to measure not only oxygen consumption rate (OCR), but also the rate of extracellular acidification (ECAR), which is a key indicator of glycolysis. The realtime measurements are carried out in multi-well plates which are provided with solid-state sensors consisting of two fluorophores. One is quenched by oxygen (O2) and the other one is sensitive to pH-value changes. The fluorophores are excited via light-emitting fiber optic bundles, which subsequently detect the fluorescence changes as a result of oxygen consumption or extracellular acidification (Plitzko and Loesgen, 2018). Furthermore, XF Analyzers enable up to four different injections per well during the measurement. All the properties mentioned constitute a significant advantage over the conventionally used clark-type oxygen electrodes for determining oxygen consumption.


Peripheral blood mononuclear cells (PBMCs) as sample in a XF Analyzer implies that the cells have to be attached to the surface of the microplates. Most commonly, this immobilization is done by means of protein solutions such as Cell-TakTM (Jones et al., 2015; Traba et al., 2016; Lee et al., 2019) or Poly-D-Lysine (Hartman et al., 2014; Nicholas et al., 2017; Thaventhiran et al., 2019). We examined both Cell-TakTM and Poly-D-Lysine and considered Poly-D-Lysine as most suitable coating method. Since we conducted the Agilent Seahorse XF Cell Mito Stress Test, optimal concentrations of the injected compounds oligomycin, FCCP, antimycin A and rotenone had to be tested as well. A typical curve of a Mito Stress Test is shown in Figure 1. Since oligomycin is an inhibitor of ATP synthase, OCR decreases after its injection. In contrast, OCR increases sharply after FCCP injection, which is an uncoupler of oxidative phosphorylation. The last injection of antimycin A and rotenone, again leads to a decline of OCR, as these two compounds inhibit complex III respectively I of the electron transport chain. The resulting curve is used to calculate various parameters of mitochondrial function (see Figure 1).



Figure 1. Assessment of mitochondrial respiration parameters by means of Agilent Seahorse XF Cell Mito Stress Test. On the left side a typical course of an OCR measurement with injections of oligomycin after the third, FCCP after the sixth and antimycin A together with rotenone after the ninth measuring point is shown. From this curve, various parameters can be calculated, which are marked in different colors. The calculations of the parameters are shown on the right side.


The protocol described in detail hereafter has been applied to examine the influence of 12 days of in vivo caloric reduction in humans. A significant increase of mitochondrial respiratory parameters could be detected in PBMCs of a subgroup of the test persons (Schöller-Mann et al., 2020). Besides such ex vivo studies the protocol can be used to screen any soluble substance, including pharmaceuticals, dietary supplements or contaminants, due to their in vitro effects on PBMCs.


Materials and Reagents

  1. 15 ml, 50 ml screw cap tubes (SARSTEDT, catalog numbers: 62.554.502; 62.547.254)

  2. 1.5 ml, 2 ml reaction tubes (SARSTEDT, catalog numbers: 72.706; 72.695.500)

  3. 20 µl, 200 µl, 1,000 µl pipette tips (SARSTEDT, catalog numbers: 70.1116; 70.760.002; 70.762)

  4. 5 ml, 10 ml, 25 ml serological pipettes (SARSTEDT, catalog numbers: 86.1253.001; 86.1254.001; 86.1685.001)

  5. 50 ml LeucosepTM tubes (Greiner Bio-One GmbH, catalog number: 227290)

  6. Seahorse XF24 FluxPak containing XF24 sensor cartridges, XF24 cell culture microplates and XF Calibrant Solution (Agilent Technologies, catalog number: 100850-001)

  7. Human venous blood, EDTA-anticoagulated – S-Monovette® 7.5 ml K3E (SARSTEDT, catalog number: 01.1605.001)

  8. Ficoll-PaqueTM PLUS (GE Healthcare, catalog number: 17-1440-03)

  9. Poly-D-Lysine solution, 1.0 mg/ml (Merck Millipore, catalog number: A-003-E, storage temp. -20 °C)

  10. Trypan Blue solution 0.4% (Sigma-Aldrich, catalog number: 93595)

  11. Dulbecco’s Modified Eagle’s Medium (DMEM), high glucose (Sigma-Aldrich, catalog number: D7777, storage temp. 4 °C)

  12. Dimethyl sulfoxide (DMSO) (Carl Roth, catalog number: A994)

  13. 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

  14. Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; Sigma-Aldrich, catalog number: C2920, storage temp. 4 °C), 2.5 mM in DMSO

  15. Rotenone (Sigma-Aldrich, catalog number: R8875), 2.5 mM in DMSO

  16. Antimycin A from Streptomyces sp. (Sigma-Aldrich, catalog number: A8674, storage temp. -20 °C), 2.5 mM in DMSO

  17. NaCl (Carl Roth, catalog number: 3957)

  18. NaOH (Carl Roth, catalog number: 6771)

  19. KCl (Carl Roth, catalog number: 6781)

  20. Na2HPO4·12H2O (Carl Roth, catalog number: N350)

  21. KH2PO4 (Carl Roth, catalog number: 3904)

  22. 1× PBS (see Recipes)

  23. 0.9% NaCl solution (see Recipes)

  24. 10 M NaOH solution (see Recipes)

  25. Poly-D-Lysine working solution (50 µg/ml) (see Recipes)

  26. 2.5 mM oligomycin solution (see Recipes)

  27. 2.5 mM FCCP solution (see Recipes)

  28. 0.1 M rotenone solution (see Recipes)

  29. 2.5 mM rotenone solution (see Recipes)

  30. 2.5 mM antimycin A solution (see Recipes)

  31. Assay medium (see Recipes)

Equipment

  1. Pipettes: Eppendorf Research® Plus 10 μl, 20 µl, 200 µl, 1,000 µl (Eppendorf, catalog numbers: 3123000020; 3123000039; 3123000055; 3123000063)

  2. Pipetting aid: PipetBoy acu 2 (Integra Biosciences, catalog number: 155 000)

  3. Neubauer counting chamber improved (Carl Roth, catalog number: PC72.1)

  4. Inverted microscope: Primovert (Carl Zeiss, catalog number: 415510-1100-000)

  5. Water bath (GFL, catalog number: 1003)

  6. Incubator without CO2 (GFL, catalog number: 4010)

  7. Swing out rotor centrifuge: 5804 R (Eppendorf, catalog numbers: 5805000010; 5804709004)

  8. Water purification system: ELGA® PURELAB flex 3 (Veolia, catalog number: PF3XXXXM1)

  9. pH meter: FE20 FiveEasyTM (Mettler Toledo, cataog number: 30266626)

  10. Seahorse XF24 Extracellular Flux Analyzer (Agilent Technologies, catalog number: 100737-100)

Software

  1. Wave Controller Software (Agilent Technologies, version 1.8.1.1)

  2. 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


  1. 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


  2. 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-PaqueTM PLUS (RT) into a 50 ml LeucosepTM 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 LeucosepTM 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 LeucosepTM 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 LeucosepTM tube. On the left side, the tube is filled with Ficoll-PaqueTM 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-PaqueTM – porous barrier – Ficoll-PaqueTM – 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 CO2 until 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

  1. 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


  2. 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.

  3. 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.

  4. 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

  1. 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.

  2. 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. 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

  2. 0.9% NaCl solution

    9 g NaCl

    ddH2O to 1 L

  3. 10 M NaOH solution

    40 g NaOH

    ddH2O to 100 ml

  4. Poly-D-Lysine working solution (50 µg/ml)

    50 µl Poly-D-Lysine solution, 1.0 mg/ml

    950 µl ddH2O

  5. 2.5 mM oligomycin solution

    5 mg oligomycin

    2.528 ml DMSO

  6. 2.5 mM FCCP solution

    10 mg FCCP

    15.737 ml DMSO

  7. 0.1 M rotenone solution

    1 g rotenone

    25.353 ml DMSO

  8. 2.5 mM rotenone solution

    25 µl of 0.1 M rotenone solution

    975 µl DMSO

  9. 2.5 mM antimycin A solution

    25 mg antimycin A

    18.529 ml DMSO

  10. 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.

References

  1. Akbari, M., Kirkwood, T. B. L. and Bohr, V. A. (2019). Mitochondria in the signaling pathways that control longevity and health span. Ageing Res Rev 54: 100940.
  2. Chaban, Y., Boekema, E. J. and Dudkina, N. V. (2014). Structures of mitochondrial oxidative phosphorylation supercomplexes and mechanisms for their stabilisation. Biochim Biophys Acta 1837(4): 418-426.
  3. Hartman, M. L., Shirihai, O. S., Holbrook, M., Xu, G., Kocherla, M., Shah, A., Fetterman, J. L., Kluge, M. A., Frame, A. A., Hamburg, N. M. and Vita, J. A. (2014). Relation of mitochondrial oxygen consumption in peripheral blood mononuclear cells to vascular function in type 2 diabetes mellitus. Vasc Med 19(1): 67-74.
  4. Jones, N., Piasecka, J., Bryant, A. H., Jones, R. H., Skibinski, D. O., Francis, N. J. and Thornton, C. A. (2015). Bioenergetic analysis of human peripheral blood mononuclear cells. Clin Exp Immunol 182(1): 69-80.
  5. Lee, H. T., Lin, C. S., Pan, S. C., Wu, T. H., Lee, C. S., Chang, D. M., Tsai, C. Y. and Wei, Y. H. (2019). Alterations of oxygen consumption and extracellular acidification rates by glutamine in PBMCs of SLE patients. Mitochondrion 44: 65-74.
  6. Matt, K. and Bergemann, J. (2019). Ex vivo Analysis of DNA Repair Capacity of Human Peripheral Blood Mononuclear Cells by a Modified Host Cell Reactivation Assay. Bio-protocol 9 (15): e3325.
  7. Nicholas, D., Proctor, E. A., Raval, F. M., Ip, B. C., Habib, C., Ritou, E., Grammatopoulos, T. N., Steenkamp, D., Dooms, H., Apovian, C. M., Lauffenburger, D. A. and Nikolajczyk, B. S. (2017). Advances in the quantification of mitochondrial function in primary human immune cells through extracellular flux analysis. PLoS One 12(2): e0170975.
  8. Pfanner, N., Warscheid, B. and Wiedemann, N. (2019). Mitochondrial proteins: from biogenesis to functional networks. Nat Rev Mol Cell Biol 20(5): 267-284.
  9. Plitzko, B. and Loesgen, S. (2018). Measurement of Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in Culture Cells for Assessment of the Energy Metabolism. Bio-protocol 8(10): e2850.
  10. Schöller-Mann, A., Matt, K., Schniertshauer, D., Hochecker, B. and Bergemann, J. (2020). 12 days of in vivo caloric reduction can improve important parameters of aging in humans. Mech Ageing Dev 188: 111238.
  11. Smolina, N., Bruton, J., Kostareva, A. and Sejersen, T. (2017). Assaying Mitochondrial Respiration as an Indicator of Cellular Metabolism and Fitness. Methods Mol Biol 1601: 79-87.
  12. Thaventhiran, T., Wong, W., Alghanem, A. F., Alhumeed, N., Aljasir, M. A., Ramsey, S., Sethu, S., Yeang, H. X. A., Chadwick, A. E., Cross, M., Webb, S. D., Djouhri, L., Ball, C., Stebbings, R. and Sathish, J. G. (2019). CD28 Superagonistic Activation of T Cells Induces a Tumor Cell-Like Metabolic Program. Monoclon Antib Immunodiagn Immunother 38(2): 60-69.
  13. Traba, J., Miozzo, P., Akkaya, B., Pierce, S. K. and Akkaya, M. (2016). An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes. J Vis Exp(117). DOI: 10.3791/54918.


简介

[摘要]正如我们今天所知,细胞的健康和功能在很大程度上取决于线粒体的功能。线粒体的基本功能是ENER GY生产,更精确的LY ATP生产,通过氧化磷酸化。因此,线粒体能量产生参数代表重要的生物标记。对人类细胞的研究主要是在体外细胞培养中进行的。然而,外周血单核细胞(PBMC)是特别升ý适于这样的检查。这就是为什么这个协议描述测量与最新的技术,新鲜分离的PBMC线粒体功能的关键参数的方法的XF分析。对于这个离体PBMC首先是从人抗凝血液中分离出来的。接下来,将它们附着到预先涂有Poly-D-Lysine的特殊微孔板的表面上。期间的氧消耗速率(OCR)以及细胞外酸化率(ECAR)的应力试剂寡,羰氰化物随后的测量4 - (三氟甲氧基)苯腙(FCCP),鱼藤酮和抗霉素A被注入。可以从获得的结果中计算出几个线粒体参数。该协议的应用允许分析对人体细胞的各种影响,例如药物或环境因素。


[背景]线粒体在维持正常细胞功能中起关键作用。现在众所周知,它们不仅通过氧化磷酸化产生ATP,而且还参与氨基酸,脂质和核苷酸的代谢,潜水信号转导和氧化还原过程以及质量控制和降解过程,包括线粒体和磷酸化。细胞凋亡(Pfanner等,2019)。然而,线粒体代表正常细胞中ATP合成的主要位点(Akbari等人,2019)。为此,通过多亚基酶复合物I – IV在线粒体内膜上产生电化学质子梯度。ATP合酶(也称为复合物V)使用此质子梯度将ADP转变为ATP (Chaban等人,2014)。

氧化磷酸化的过程与氧气还原成水有关。因此,细胞的耗氧率可用于评估线粒体功能(Smolina等人,2017)。该原理是Seahorse XF分析仪(Agilent Technologies)的基础。它们不仅可以测量耗氧率(OCR),而且可以测量胞外酸化率(EC AR),这是糖酵解的关键指标。实时测量在多孔板中进行,多孔板配备有由两个荧光团组成的固态传感器。一种通过氧气(O 2 )淬灭,另一种对pH值变化敏感。荧光团通过发光的光纤束激发,随后检测到由于耗氧或细胞外酸化引起的荧光变化(Plitzko和Loesgen,2018)。此外,XF分析仪在测量过程中每孔最多可以进行四次不同的进样。所提及的所有特性构成了与常规用于确定氧气消耗量的克拉克型氧气电极相比的显着优势。

在XF分析仪中作为样本的外周血单核细胞(PBMC)意味着这些细胞必须附着在微孔板的表面。最常见的是,这种固定是通过诸如Cell-Tak TM (Jones等人,2015; Traba等人,2016 ; Lee等人,2019)或Poly-D-Lysin e (Hartman等人)的蛋白质溶液完成的。等人,2014;Nicholas等人,2017; Thaventhiran等人,2019)。我们检查了Cell-Tak TM和Poly-D-Lysin e,并认为Poly-D-Lysin e是最合适的包衣方法。由于我们进行了安捷伦Seahorse XF细胞线粒体压力测试,因此还必须测试注射化合物寡聚霉素,FCCP,抗霉素A和鱼藤酮的最佳浓度。一个三刀应力测试中示出的典型曲线˚F igure 1.由于寡是ATP合酶的抑制剂,其OCR注射后减小。相反,FCCP注射后OCR急剧增加,这是氧化磷酸化的解偶联剂。最后一次注射抗霉素A和鱼藤酮会再次导致OCR下降,因为这两种化合物分别抑制电子传输链的复合物III和I。将得到的曲线是用来计算线粒体功能的各种参数(见˚F igure 1)。





图1.通过Agilent Seahorse XF细胞线粒体压力测试评估线粒体呼吸参数。左侧显示了典型的OCR测量过程,在第三次测量后注射寡霉素,第六次后注射FCCP,在第九个测量点后注射鱼藤酮和鱼藤酮。从该曲线可以计算出各种参数,这些参数以不同的颜色标记。参数的计算显示在右侧。



该协议将在后面详细描述已被应用于吨ö检查的12天的影响在体内的热量减少在人类。在测试人员亚组的PBMC中可以检测到线粒体呼吸参数的显着增加(Schöller-Mann等人,2020)。除了此类离体研究以外,由于其对PBMC的体外影响,该方案还可用于筛选任何可溶性物质,包括药物,膳食补充剂或污染物。

关键字:人外周血单核细胞, 线粒体, XF 分析仪, XF线粒体压力测试, 耗氧率, 线粒体呼吸, 体外



材料和试剂


1. 15 ml,50 ml螺帽管(SARSTEDT,目录号:62.554.502 ;62.547.254)     

2. 1.5毫升,2毫升反应管(SARSTEDT,目录号:72.706 ;72.695.500)     

3. 20微升,200微升,1 ,000微升移液管尖端(SARSTEDT,产品目录号:70.1116 ; 70.760.002 ; 70.762)     

4. 5毫升,10毫升,25毫升血清移液管(SARSTEDT,目录号:86.1253.001 ; 86.1254.001 ; 86.1685.001)     

5. 50毫升Leucosep TM管小号(格雷纳生物一个GmbH的,目录号:227290)     

6.包含XF24传感器盒,XF24细胞培养微孔板和XF校准溶液的Seahorse XF24 FluxPak(Agilent Technologies,目录号:100850-001)     

7.人静脉血,EDTA抗凝- S-Monovette ® 7.5毫升K3E(SARSTEDT,目录号:01.1605.001)     

8. Ficoll-Paque TM PLUS(GE Healthcare,目录号:17-1440-03)     

9.聚-D-赖氨酸溶液,1.0 mg / ml(默克密理博,目录号:A-003-E,储存温度-20°C)     

10.台盼蓝溶液0.4%(Sigma-Aldrich,目录号:93595) 

11. Dulbecco改良的Eagle's培养基(DMEM),高葡萄糖(Sigma-Aldrich,目录号:D7777,储存温度4°C) 

12.二甲基亚砜È (DMSO)(卡尔罗斯,目录号:A994) 

13.从寡霉素链霉omyces diastatochromogenes (Sigma-Aldrich公司,目录号:O4876,储存温度-20℃;异构体A,B,和C的混合物。),2.5mM的在DMSO中 

14.羰基氰化物4-(三氟甲氧基)苯hydr (FCCP; Sigma-Aldrich,目录号:C2920,存储温度4°C),在DMSO中为2.5 mM 

15.鱼藤酮(Sigma-Aldrich,目录号:R8875),在DMSO中为2.5 mM 

16.来自链霉菌属的抗霉素A。(Sigma-Aldrich,目录号:A8674,储存温度-20°C),在DMSO中为2.5 mM 

17. NaCl(C arl Roth,目录号:3957) 

18. NaOH(C arl Roth,目录号:6771) 

19. KCl(卡尔·罗斯,目录号:6781) 

20. Na 2 HPO 4 ·12H 2 O(卡尔·罗斯,目录号:N350) 

21. KH 2 PO 4 (Carl Roth,目录号:3904) 

22. 1 × PBS(请参阅食谱) 

23. 0.9%NaCl溶液(请参阅食谱) 

24. 10 M NaOH溶液(请参阅配方) 

25.聚-D-赖氨酸工作溶液(50 µg / ml)(请参见食谱) 

26. 2.5 mM寡霉素溶液(请参阅食谱) 

27. 2.5 mM FCCP解决方案(请参阅食谱) 

28. 0.1 M鱼藤酮溶液(请参阅食谱) 

29. 2.5 mM鱼藤酮溶液(请参阅食谱) 

30. 2.5 mM抗霉素A溶液(请参阅食谱) 

31.分析介质(请参见食谱) 



设备


移液器:的Eppendorf研究®加10微升,20微升,200微升,1000微升仪(Eppendorf,产品目录号:3123000020 ; 3123000039 ; 3123000055 ; 3123000063)
移液辅助工具:PipetBoy acu 2(Integra Biosciences,目录号:155 000)
改进Neubauer计数室(Carl Roth,目录号:PC72.1)
倒置显微镜:Primovert(Carl Zeiss,目录号:415510-1100-000)
水浴(GFL,目录号:1003)
不带CO 2的培养箱(GFL,目录号:4010)
摆出转子离心机:5804 R(Eppendorf,产品目录编号:5805000010; 5804709004)
水净化系统:ELGA ® PURELAB挠曲3(威立雅,目录号:PF3XXXXM1)
pH计:FE20 FiveEasy TM (Mettler Toledo,目录号:30266626)
Seahorse XF24细胞外流量分析仪(Agilent Technologies,目录号:100737-100)


软件


波形控制器软件(安捷伦技术,版本1.8.1.1)
可选:Wave桌面软件(安捷伦科技公司)


程序


用于检测在使用XF分析器人PBMC线粒体功能的实验程序示意地示于˚F igure 2.这之后的各个工作步骤的详细描述。


                         

图2.使用XF分析仪测量人PBMC中线粒体功能的工作流程图。


实验前一天
的水合XF24传感器盒(由盒盖,传感器盒本身和效用的板;参见˚F igure 3B和3C):
将1 ml XF校准溶液添加到效用板的每个孔中。
用移液器吸头除去可能在传感器上产生的气泡。
将组装好的板在非CO 2 37°C的培养箱中孵育过夜。
交换机上的XF24分析仪(见˚F igure 3A)一个第二让它升温至37℃过夜。




图3. XF24分析仪(A)和XF24传感器盒(B和C)。从上方(B)和下方(C)显示了包括传感器标记的传感器盒。此外,注油口标有(B),并显示了效用板(C)。


在XF分析仪上编程测量协议:
平衡:30分钟
测量周期1:
混合:4分钟
等待:2分钟
测量:3分钟
3次重复

端口A的喷射(50微升的7.5μM寡霉素;终浓度0.75μM)
测量期2:
混合:4分钟
等待:2分钟
测量:3分钟
3次重复

端口B的注射(55微升的10μM FCCP ;终浓度1μM)
测量期3:
混合:4分钟
等待:2分钟
测量:3分钟
3次重复

端口C的注射(60微升的16.7μM鱼藤酮/抗霉素A;终浓度1.67μM)
测量周期4:
混合:4分钟
等待:2分钟
测量:3分钟
3次重复


实验日
XF24细胞培养微孔板的涂层:
向每个孔中加入30 µl的Poly-D-赖氨酸工作溶液(50 µg / ml),并将板在室温下孵育1小时。
弃去上清液,用300 µl ddH 2 O洗涤每孔,并在无菌条件下(约30分钟)将平板风干。
的PBMC的分离(继续与微孔板涂层在该步骤期间;见˚F igure 4 ):
将15 ml的Ficoll-Paque TM P LUS(RT)加入50 ml的Leucosep TM试管中,并在室温以1,000 × g的浓度离心30 s。
用0.9%NaCl溶液(至少7.5 ml和最多15 ml)以1:2的比例稀释适当体积的抗凝人体血液。
将所得的体积(至少15 ml,至多30 ml)转移到准备好的Leucosep TM管中,并在不带制动器的摆出式转子中于室温以1,000 × g离心10分钟。
收获PBMC馏分,该血浆在Leucosep TM管的多孔屏障上方的血浆和Ficoll之间显示为白色层(有关详细信息,请参见制造商的说明或参阅Matt and Bergemann,(2019 ))。为此,请使用1,000 µl Eppendorf移液器,并将PBMC收集在50 ml螺帽管中。
用10 ml PBS洗涤细胞一次,并以250 × g离心10分钟。
重复洗涤步骤两次,每次5 ml PBS。
使用改进的Neubauer计数室确定细胞数。             




图4.用Leucosep TM管分离PBMC 。在左侧,该试管的下方装有Ficoll-Paque TM ,而在多孔屏障上方则装有抗凝和稀释的血液。在右侧,密度梯度离心后,试管从上到下显示以下几层:血浆– PBMC – Ficoll-Paque TM –多孔屏障– Ficoll-Paque TM –红细胞和粒细胞。


使用前准备好测定介质并在37°C下预热。
播种的PBMC ONT ö预涂覆XF24细胞培养微板中:
注意:每种条件下建议使用4至5口井。A1,B4,C3和D6孔通常用作背景孔(无细胞)。一种示例性的板布局以及接种在聚d赖氨酸的PBMC的图像中示出˚F igure 5。

每孔取5 × 10 5分离的PBMC,并以250 × g离心5分钟。
每孔将细胞沉淀重悬于100 µl分析培养基中。
每孔添加100 µl细胞悬液。
用450 µl分析培养基填充背景孔。
在平开的转子中以250 × g的速度将板离心1分钟,并关闭制动器。
向装有细胞的孔中加入350 µl测定培养基(每孔的最终体积:450 µl)。
在无CO 2的情况下于37°C孵育平板直至测量(平均约1 h )。




图5. XF24细胞培养微孔板的示例性板布局(左侧)和接种在Poly-D-赖氨酸涂层的XF24细胞培养微孔板(右侧)上的PBMC的明场显微镜图像。左侧:Ť他孔A1,B4,C3和D6被用作背景孔(无细胞)。一种颜色(浅绿色,深绿色,深橙色,浅橙色)分别代表一种状态。每个条件有5口井。右侧:图像以100 ×放大倍数显示PBMC 。


在XF24传感器盒中装入化合物:
制备化合物如下:
稀4.86微升的2.5mM的寡霉素溶液在1615微升测定培养基(导致7.5μM)。  
稀6.48微升的2.5mM的FCCP溶液在1614微升测定培养基(导致10μM)。             
在1,598 µl分析培养基中稀释10.82 µl的2.5 mM鱼藤酮溶液和10.82 µl的2.5 mM抗霉素A溶液(每次结果16.7 µM)。
如下装入XF24传感器盒的进样口:
注意:避免在进样口中形成气泡,并在装入后小心操作XF24传感器盒,以防止化合物滴落。

填充所有端口A与50μl的7.5μM寡霉素。
用55微升填充所有端口乙的10μMFCCP。
填写所有端口下用60微升的16.7μM鱼藤酮/抗霉素A.
在XF分析仪中开始运行(按照软件说明进行操作):
将XF24传感器盒(无盖)放入XF分析仪中,然后开始校准。
校准完成后,用XF24细胞培养微孔板替换效用板并开始测量。
将测量结果归一化为细胞数:
通过反复上下移液,将PBMC重悬在每个孔的最终体积(615 µl)中。
使用改进的Neubauer计数室确定每孔的细胞数。             






数据分析


通过如图10所示,划分每孔的细胞的计数数目000,并且为了normali进入下“正常化”波浪软件结果泽的数据(见˚F igure 6)。




图6. Wave软件的屏幕截图,其中带有用于规范化和数据导出的标记。


根据比较一种情况的单井的OCR进程来定义异常值,并将其排除在分析之外。
注意:至少应使用三个重复进行分析。

结果导出到海马XF细胞美图压力测试报告生成器,它链接到浪潮软件(见˚F igure 6)。该软件工具会自动计算并报告参数,然后由Mito Stress Test进行分析。
从报告生成器中获取计算出的值,并使用适当的统计软件对其进行分析。


代表数据

使用XF分析仪和应力试剂寡,FCCP,抗霉素A的氧消耗率测量的典型结果和鱼藤酮(三刀应力测试)的所示˚F igure 7。




图7.用XF分析仪测量的PBMC的Mito压力测试的耗氧率(OCR)。描绘了两个不同主题的结果未归一化(A)和归一化(B)。






笔记


由于人体血液样本通常只能在每种情况下获得一次,因此建议对冷冻保存的标准PBMC样本进行测量。这可以用于其他标准化步骤。
所述的端口d XF24传感器盒没有在所描述的实验中使用并为此Ë所有端口d可以留空。在端口A,B和C中,必须填充所有24口井,以确保均匀注入化合物。


菜谱


1 × PBS
8克氯化钠

0.20克氯化钾

2.88 g Na 2 HPO 4 ·12H 2 O

1.24克KH 2 PO 4

ddH 2 O至1 L,pH 7.4

0 。9%NaCl溶液
9克氯化钠

ddH 2 O至1 L

10 M NaOH溶液
40克NaOH

ddH 2 O至100 ml

聚-D-赖氨酸工作溶液(50 µg / ml)
50 µl聚-D-赖氨酸溶液,1.0 mg / ml

950微升ddH 2 O

2.5 mM寡霉素溶液
5毫克寡霉素

2.528毫升DMSO

2.5 mM FCCP解决方案
10毫克FCCP

15.737毫升DMSO

0 。1 M鱼藤酮溶液
1克鱼藤酮

25.353毫升DMSO

2.5 mM鱼藤酮溶液
25微升的0.1M的鱼藤酮溶液

975微升DMSO

2.5 mM抗霉素A溶液
25毫克抗霉素A

18.529毫升DMSO

测定培养基
0.675克Dulbecco改良的Eagle's中高葡萄糖

ddH 2 O至50 ml,pH 7.4


致谢


这项工作是由巴登-符腾堡州科学,研究和艺术部通过“合作研究生院InViTe”资助的。我们要感谢med博士。Adrian Schulte和他在Überlingen-Hödingen的F. X. Mayr Bodensee中心的团队提供了血液样本。


利益争夺


作者宣称没有利益冲突。


伦理


实验根据赫尔辛基宣言进行,并由德国LandesärztekammerBaden-Württemberg伦理委员会批准。此外,所有志愿者均被提前告知,并书面同意使用其血样。


参考


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引用:Schöller-Mann, A., Matt, K., Hochecker, B. and Bergemann, J. (2021). Ex vivo Assessment of Mitochondrial Function in Human Peripheral Blood Mononuclear Cells Using XF Analyzer. Bio-protocol 11(7): e3980. DOI: 10.21769/BioProtoc.3980.
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