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Oct 2017

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Measurement of Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in Culture Cells for Assessment of the Energy Metabolism
测定培养细胞中的消氧率（OCR）和细胞外酸化率（ECAR）以评估能量代谢   

Birte PlitzkoBirte Plitzko Sandra LoesgenSandra Loesgen
DOI:   10.21769/BioProtoc.2850
卷期号： Vol 8, Iss 10, May 20, 2018
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Abstract

Mammalian cells generate ATP by mitochondrial (oxidative phosphorylation) and non-mitochondrial (glycolysis) metabolism. Cancer cells are known to reprogram their metabolism using different strategies to meet energetic and anabolic needs (Koppenol et al., 2011; Zheng, 2012). Additionally, each cancer tissue has its own individual metabolic features. Mitochondria not only play a key role in energy metabolism but also in cell cycle regulation of cells. Therefore, mitochondria have emerged as a potential target for anticancer therapy since they are structurally and functionally different from their non-cancerous counterparts (D'Souza et al., 2011). We detail a protocol for measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements in living cells, utilizing the Seahorse XF24 Extracellular Flux Analyzer (Figure 1). The Seahorse XF24 Extracellular Flux Analyzer continuously measures oxygen concentration and proton flux in the cell supernatant over time (Wu et al., 2007). These measurements are converted in OCR and ECAR values and enable a direct quantification of mitochondrial respiration and glycolysis. With this protocol, we sought to assess basal mitochondrial function and mitochondrial stress of three different cancer cell lines in response to the cytotoxic test lead compound mensacarcin in order to investigate its mechanism of action. Cells were plated in XF24 cell culture plates and maintained for 24 h. Prior to analysis, the culture media was replaced with unbuffered DMEM pH 7.4 and cells were then allowed to equilibrate in a non-CO2 incubator immediately before metabolic flux analysis using the Seahorse XF to allow for precise measurements of Milli-pH unit changes. OCR and ECAR were measured under basal conditions and after injection of compounds through drug injection ports. With the described protocol we assess the basic energy metabolism profiles of the three cell lines as well as key parameters of mitochondrial function in response to our test compound and by sequential addition of mitochondria perturbing agents oligomycin, FCCP and rotenone/antimycin A.


Figure 1. Overview of seahorse experiment

Keywords: Bioenergetics (生物能量学), Seahorse XF (海马XF), Mitochondrial metabolism (线粒体代谢), Glycolysis (糖酵解), Mitochondrial respiration (线粒体呼吸)

Background

Natural products are small molecules that are isolated from natural sources. Over the last century, these molecules have been instrumental in treating human diseases, especially inspired chemotherapeutics. Metabolites like taxol, vincristine, and doxorubicin have revolutionized how we treat malign cancers and other natural products, for example rapamycin, oligomycin, and bafilomycin, are used as molecular probes and enable molecular studies of biochemical and cellular processes in the laboratory. While studying the mechanism of action of the cytotoxic natural product mensacarcin, we found that a fluorescently labeled mensacarcin probe localizes to a great extent in mitochondria (Plitzko et al., 2017). To investigate if mensacarcin’s cytotoxic properties might be derived from interference with mitochondrial function, we sought to examine mensacarcin’s effects on cellular bioenergetics. Using a Seahorse Extracellular Flux Analyzer, we monitored cellular oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) in real time as measures of mitochondrial respiration and glycolysis, respectively (Wu et al., 2007; Serill et al., 2015). The Seahorse XF24 Extracellular Flux Analyzer allows continuous direct quantification of mitochondrial respiration and glycolysis of living cells. The instrument uses a sensor cartridge in a 24-well plate format with each sensor being equipped with two embedded fluorophores: one which is quenched by oxygen (O2) and the other that is sensitive to change in pH. During measurements, the sensor cartridge is lowered 200 µm above the cell monolayer, forming a micro-chamber of about 2 µl. The Seahorse instrument contains fiber optic bundles that emit light, excite the fluorophores, and then measures the change in the fluorophore’s emission. The very small test volume formed by the transient micro chamber allows for sensitive, precise, and nondestructive measurements of parameters in real time. Changes in oxygen concentration and pH are automatically calculated and reported as Oxygen Consumption Rate (OCR) and Extra Cellular Acidification Rate (ECAR). Once a measurement is completed, the sensors lift which allows the larger medium volume above to mix with the medium in the transient micro chamber, restoring values to baseline. The sensor cartridge contains ports that allow sequential addition of up to four compounds per well during the assay measurements.

With the described protocol we assessed the energy metabolism of three cell lines (HCT-116, SK-Mel-28, and SK-Mel-5) (Figure 6). Addition of mensacarcin was found to have pronounced effect on the basal OCR of melanoma cells and no increasing effect on ECAR. An increase in glycolysis is often observed as a compensatory response. Mitochondria are essential for the energy metabolism of cells and have a key role in apoptotic cell death. Alteration of the mitochondrial respiration or the equilibrium between the pro-apoptotic and anti-apoptotic proteins can induce mitochondrial failure. To gain insights into the induced mitochondrial impairment in melanoma cells, we assessed key parameters of mitochondrial respiration by consecutively exposing cells to well described mitochondria perturbing reagents. Following addition of our test compound mensacarcin, we sequentially added oligomycin, FCCP, and lastly rotenone and antimycin A (Figure 5). Oligomycin inhibits ATP synthase and reduces OCR, FCCP uncouples oxygen consumption from ATP production and raises OCR to a maximal value, and antimycin A and rotenone target the electron transport chain and reduce OCR to a minimal value. The mitochondria stress test protocol provides information on basal respiration, ATP-linked respiration, proton leak, maximal respiration capacity, and non-mitochondrial respiration of cells. Therefore, this assay can be used to provide insight on the mechanism of action of compounds that directly target mitochondrial respiration.

Traditional measurements of mitochondrial function or glycolysis require an oxygen electrode, or kits and dyes that utilize colorimetric or fluorimetric detection (Li and Graham, 2012; TeSlaa and Teitell, 2014). Most of these methods are invasive and cumbersome methods that only allow low sample throughput. In contrast, the Seahorse analyzer assay with its sensor cartridge system enables measurement of mitochondrial respiration and glycolysis in real time and in a non-invasive manner that does not require any dyes or labels. Cellular energy metabolism research is highly topical in all fields of mammalian cell biology. The following protocol was developed for researchers in cancer biology but with approaches that suit studies of energy metabolism in all mammalian cell systems.

Materials and Reagents

  1. CELLSTAR® Tissue Culture Plates, 96-well (Greiner Bio One International, catalog number: 655180 )
  2. Sterile racked pipette tips (1 ml and 200 μl) (VWR, catalog numbers: 613-0738 ; 613-0742 )
  3. Sterile basins (Corning, Costar®, catalog number: 4870 )
  4. Sterile reagent tubes (15 and 50 ml) (VWR, catalog numbers: 89039-668 ; 89039-662 )
  5. Sterile Serological pipettes (5, 10, 25, 50 ml) (Fisher Scientific, catalog numbers: 13-678-11 , 13-678-11D , 13-678-11E , 13-678-11F )
  6. Glass bottles (500 ml) (Fisher Scientific, catalog number: FB8001000 )
  7. HCT-116, SK-Mel-5 and SK-Mel-28 cells (ATCC, catalog numbers: CCL-247 , HTB-70 , HTB-72 )
  8. Seahorse XF24 FluxPak (including sensor cartridges, tissue culture plates, calibrant solution and calibration plates) (Agilent Technologies, Santa Clara, CA)
  9. Trypsin/EDTA (0.25%/2.21 mM) (Corning, catalog number: 25-053-Cl )
  10. 1x Ca2+/Mg2+-free DPBS (Thermo Fisher Scientific, GibcoTM, catalog number: 14190250 )
  11. Liquid Dulbecco’s modified Eagle’s medium (DMEM) (Corning, catalog number: 10-013 )
  12. Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S11150 )
  13. Penicillin/streptomycin solution 100x (Corning, catalog number: 30-002-Cl )
  14. Powder Dulbecco’s modified Eagle’s medium (DMEM) without Na2HCO3, without HEPES (Corning, catalog number: 50-013 )
  15. Sodium hydroxide (NaOH) (VWR, catalog number: 97064-476 )
  16. Oligomycin (Merck, catalog number: 495455-10MG )
  17. DMSO (VWR, catalog number: BDH1115-1LP )
  18. FCCP (Cayman Chemical, catalog number: 15218 )
  19. Rotenone (Cayman Chemical, catalog number: 13995 )
  20. Antimycin A (Enzo Life Sciences, catalog number: ALX-380-075-M005 )
  21. Culture media (10% (v/v) FBS) (see Recipes)
  22. Assay media (see Recipes)
  23. NaOH (1 M) (see Recipes)
  24. Oligomycin (10 µM) (see Recipes)
  25. FCCP (5 µM) (see Recipes)
  26. Rotenone (5 µM)/antimycin A (5 µM) (see Recipes)

Equipment

  1. Hemacytometer (Hausser Scientific, catalog number: 1490 )
  2. Biological Safety Cabinet Class II, Type A2 (NuAire, model: NU-425-400ES )
  3. Seahorse XF Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA)
  4. Pipet-Lite Pipette XLS STD 20 XLS (Mettler Toledo, Rainin, model: SL-2XLS+ )
  5. Pipet-Lite Pipette XLS STD 200 (Mettler Toledo, Rainin, model: SL-200XLS+ )
  6. Pipet-Lite Pipette XLS 1000 (Mettler Toledo, Rainin, model: SL-1000XLS+ )
  7. Multichannel Pipet-Lite Pipette XLS 8-CH 1200 (Mettler Toledo, Rainin, model: L8-1200XLS+ )
  8. Multichannel Pipet-Lite Pipette XLS 8-CH 200 (Mettler Toledo, Rainin, model: L8-200XLS+ )
  9. Aspirator pump
  10. Humidified non-CO2 incubator (XF Prep Station; Agilent Technologies, Santa Clara, CA)
  11. Shallow water bath (Thermo Fisher Scientific, Thermo ScientificTM, model: Precision 180 )
  12. Pipette controller (BrandTech Scientific, model: Accu-Jet® Pro , catalog number: 26330)
  13. Humidified, 37 °C, 5% CO2 incubator (Eppendorf, model: Galaxy® 170 R )
  14. -20 °C biomedical freezer (Sanyo, model: MDF-U731M )
  15. Autoclave (Consolidated Sterilizer Systems, model: SSR-3A , ADVPB)
  16. Inverted light microscope (Nikon Instruments, model: Eclipse TS100 )
  17. pH-meter with semi-micro electrode (Thermo Fisher Scientific, Thermo ScientificTM, model: Orion StarTM A211 , with ROSS 8103BN electrode: (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 8103BN )

Software

  1. Seahorse Bioscience XF24 software
  2. Excel (Microsoft)
  3. GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA)

Procedure

  1. Optimization of seeding density
    In an initial experiment, the optimal seeding density is required for each cell type. Typically, the cell density ranges from 10,000 to 60,000 cells per well and can vary widely among cell lines. A first point of orientation can be the cell number that gives confluency of approx. 95% overnight in a 96-well cell culture plate as the seeding surface is comparable to the seahorse culture plate. The seeding number should give a confluent and healthy and consistent monolayer on the day of the assay.
    1. HCT-116, SK-Mel-5 and SK-Mel-28 cells were seeded in a Seahorse XF24 cell culture plate at various concentrations ranging from 10,000 to 30,000 cells/well with a two-step seeding technique as described below in Procedure B (Figure 2). Seeding cells in triplicates is recommended.


      Figure 2. Plate layout for cell density evaluation. Shown here is the exemplary seeding layout for the SK-Mel-5 and SK-Mel-28 cell lines (seeding density for HCT-116 cells was evaluated on a second plate; not shown).

    2. Cells were then assayed in the XF24 instrument as described in Procedure E (without loading compounds into ports) using Table 1 commands.

      Table 1. Protocol commands for cell density evaluation


      As seen in Figure 3, a linear increase of OCR values with increasing cell density was observed in all three cell lines. ECAR values begin to level off at 20,000 cells/well for SK-Mel-28 and SK-Mel-5 while being much lower and steadily increasing for HCT-116. Thus, a seeding number of 20,000 cells/well for SK-Mel-28 and SK-Mel-5 and of 35,000 cells/well for HCT-116 were chosen to ensure being within the linear response range while having high reading values to observe increases as well as decreases in OCR and ECAR.


      Figure 3. Optimization of assay conditions: evaluation of OCR and ECAR depending on the seeding density of three different cell lines

  2. Seeding cells into Seahorse XF24 tissue culture plate (Day 1)
    Note: The seeding and growing of cells are performed with good sterile cell culture technique. A two-step seeding method is used to obtain a consistent even monolayer which is vital to obtain consistent and accurate data:
    1. Pre-warm culture media, trypsin solution and DPBS to 37 °C.
    2. For adherent cells, wash cells with DPBS, and add trypsin and wait until cells begin to detach. Add culture media with serum to deactivate trypsin and pipette up and down to create a uniform cell suspension. Count cells with a hemocytometer and resuspend cells in growth media to the desired final concentration to seed in 100 µl.
    3. Plate 100 µl cell suspension into a Seahorse XF24 tissue culture plate. Put media only (no cells) in the background correction wells (A1, B4, C3, D6).
    4. Let the culture plate sit for 1 h in the bio-hood without moving it around (in order to let cells settle evenly).
    5. Place the culture plate into an incubator (37 °C, 5% CO2) for 4 h.
    6. Carefully add 150 µl growth media (final volume in well 250 µl). Hold the pipette tip at an angle and add to the well side to not destroy even layer of newly attached cells.
    7. Let cells grow overnight at 37 °C, 5% CO2.

Note: The following steps are performed without sterile technique, but caution to keep the cells and equipment as clean as possible. 
  1. Hydrate sensors (Day 1)
    1. Open XF 24 FluxPak and take out the sensor cartridge (green) and calibration plate (clear) (Figure 4).


      Figure 4. Seahorse XF 24 sensor cartridge. A. The sensor cartridge sitting on top of a calibration plate with injection ports shown. B. Bottom side of the sensor plate which shows sensors with embedded fluorophores.

    2. Place the sensor cartridge (sensors up) next to the calibration plate (be careful not to touch sensors).
    3. Fill each well of the calibration plate with 1 ml of Seahorse XF Calibrant.
    4. Lower the sensor cartridge onto the calibrant plate submerging the sensors in calibrant (be careful not to touch walls with sensors).
    5. Place in a non-CO2 37 °C incubator overnight. To prevent evaporation of the XF Calibrant, verify that the incubator is properly humidified.

  2. Stabilization of instrument (Day 1)
    1. Turn on an XF24 Analyzer, open Seahorse Bioscience software and log in.
    2. Write the assay template. When planning and writing the assay protocol be careful not to create a protocol that is longer than cells can manage without CO2 in unbuffered media. Depending on cell type this is 2-3 h. If in doubt, a cell viability assay can be performed after the seahorse assay.
    3. Leave the XF24 Analyzer on overnight with XF24 software running and logged in to ensure equilibration to 37 °C.

  3. Seahorse assay (Day 2)
    1. Check on the confluency of cells. Evenly spacing of cells is needed, without large cell clumps or blank patches, as this could impair the accuracy of data.
    2. Pre-warm assay media to 37 °C.
    3. Pre-warm compounds and adjust to pH 7.4 with NaOH (1 M) if necessary.
    4. Perform media exchange in a Seahorse XF24 tissue culture plate:
      1. Remove 150 µl growth media with a multichannel pipet.
      2. Add 1 ml assay media with a multichannel pipette.
      3. Remove 1 ml with a multichannel pipette.
      4. Add 475 µl assay media with a multichannel pipette (575 µl final volume).
      5. Place the cell plate into a CO2-free incubator for approx. 60 min.
    5. Load cartridge with desired compounds:
      1. Pre-warm compounds to 37 °C.
      2. Load 50-100 µl of compound into appropriate port of cartridge (for mitochondrial stress test: 64 µl into port A, 71 µl port B, 79 µl port C, 88 µl port D). (see Note 1) Load equivalent amounts of assay media into equivalent port for background wells (see Note 2).
      3. Place back into the incubator (non-CO2) for 10 min to allow heating up to 37 °C again. Handle carefully, carry only by holding onto the calibration plate. Move as less as possible.
    6. Calibration and running seahorse assay:
      1. Load assay template in Seahorse XF24 software.
      2. Press green ‘START’ button.
      3. Make sure to load the correct protocol, the correct save directory and saving name.
      4. Press ‘START’.
      5. Load sensor cartridge with calibration plate into instrument tray (the notch goes in the front, left corner. Make sure that the plate sits correctly and flat, between all 8 tabs)
      6. Follow the instructions on the screen in order to calibrate and equilibrate sensors.
      7. Once equilibration step is done, remove the calibration plate and replace with cell culture plate.

  4. Protocol commands (mitochondria stress test, Table 2, Figure 5)

    Table 2. Protocol commands for mitochondrial stress test

    5. Data analysis

    Results were initially reviewed using the seahorse XF data viewer which automatically saves data as MS Excel (.xls) file. Graphic and statistical analyses were carried out using GraphPad Prism. The significance of observed differences of the basal bioenergetics of cell lines was evaluated by the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test. In all cases, P < 0.05 was considered to be significant. Experimental values are reported as mean ± standard deviation (Figure 5) or in a box plot (Figure 6).


    Figure 5. Mitochondrial stress test. OCR was measured after mensacarcin was injected (black arrow) in different concentrations to SK-Mel-28 cells followed by consecutive injections of oligomycin (1 μM), FCCP (0.5 μM), and antimycin A (0.5 μM)/rotenone (0.5 μM) (n = 3).


    Figure 6. Basal bioenergetic state of SK-Mel-28, SK-Mel-5 and HCT-116 cells. The basal energy metabolism of each cell line was assessed by analyzing OCR/ECAR ratios. OCR and ECAR were acquired with the same protocol as described above but without the injection of compounds. The protocol commands consisted of one loop with 8 measurements. Several separate assays were performed (n = 25).

    Notes

    1. Pipet into ports with angle, do not touch the bottom, do not tap to prevent leakage. The liquid is only held by capillary forces.
    2. It is mandatory to load ports for the background wells with assay media that contains the same concentration of DMSO as the compounds to account for any DMSO effects on cells.
    3. Once injected into the wells, compounds are diluted 1:10. This will give a final concentration of 1 µM oligomycin and 0.5 µM FCCP, rotenone and antimycin A, respectively, in the cell culture well.

    Recipes

    1. Culture media (10% (v/v) FBS)
      Note: Work under sterile conditions in a laminar flow hood.
      1. Open liquid DMEM bottle
      2. Take out 55 ml with a sterile Serological pipette and discard the liquid
      3. Add 50 ml FBS with a sterile Serological pipette
      4. Add 5 ml penicillin/streptomycin solution
      5. Store at 4 °C
    2. Assay media (sterile, unbuffered, 250 ml)
      Note: Work under sterile conditions in a laminar flow hood.
      1. Autoclave 250 ml ultrapure H2O in a glass bottle
      2. Dissolve 3,34 g powder DMEM without NaHCO3 and without HEPES in 250 ml autoclaved H2O
      3. Warm to 37 °C
      4. Adjust to pH 7.40 with NaOH (1 M)
      5. Store at 4 °C
    3. NaOH (1 M)
      Dissolve 4 g NaOH pellets in 100 ml autoclaved H2O
    4. Oligomycin (10 µM)
      1. Prepare freshly on the day of seahorse assay (day 2) (see Note 1)
      2. Prepare 1 mM solution in 1 ml DMSO: Dissolve 0.7911 mg oligomycin in DMSO
      3. Dilute to 10 µM in assay media (1% DMSO): Pipet 20 µl of 1 mM oligomycin into 1,980 µl assay media
      4. Warm to 37 °C and adjust to pH 7.4 with NaOH (1 M) if necessary
    5. FCCP (5 µM)
      1. Prepare freshly on the day of seahorse assay (day 2) (see Note 1)
      2. Prepare 50 mM solution in DMSO: Dissolve 2.54 mg FCCP in 200 µl DMSO
      3. Dilute to 500 µM: Pipet 10 µl of 50 mM FCCP into 990 µl DMSO
      4. Dilute to 5 µM in assay media (1% DMSO): Pipet 20 µl of 500 µM FCCP into 1,980 µl assay media
      5. Warm to 37 °C and adjust to pH 7.4 with NaOH (1 M) if necessary
    6. Rotenone (5 µM)/antimycin A (5 µM)
      1. Prepare freshly on the day of seahorse assay (day 2) (see Note 1)
      2. Prepare 50 mM solution in DMSO: Solve 3.94 mg rotenone and 5.49 mg antimycin A in 200 µl DMSO
      3. Dilute to 1 mM: Pipet 10 µl of 50 mM rotenone/antimycin A into 490 µl DMSO
      4. Dilute to 5 µM in assay media (0.5% DMSO): Pipet 20 µl of 1 mM rotenone/antimycin A into 1,980 µl assay media
      5. Warm to 37 °C and adjust to pH 7.4 with NaOH (1 M) if necessary

    Acknowledgments

    The work was primarily supported by OSU startup funds. B.P. thanks DFG (Deutsche Forschungsgemeinschaft) for postdoctoral funding (PL 799/1-1). We wish to thank Dr. Napur Pande for providing SK-Mel-28 cells, and Bioviotica (Prof. Dr. Axel Zeeck and Hans-Peter Kroll) for providing mensacarcin. We like to thank Elizabeth N. Kaweesa for help with this protocol and Dr. Jeffrey D. Serrill and Prof. Jane E. Ishmael for information and feedback on the seahorse experiments. The authors declare that there are no conflicts of interest or competing interests.

    References

    1. D’Souza, G. G., Wagle, M. A., Saxena, V. and Shah, A. (2011). Approaches for targeting mitochondria in cancer therapy. Biochim Biophys Acta 1807(6): 689-696.
    2. Koppenol, W. H., Bounds, P. L. and Dang, C. V. (2011). Otto Warburg's contributions to current concepts of cancer metabolism. Nat Rev Cancer 11(5): 325-337.
    3. Li, Z. and Graham, B. H. (2012). Measurement of mitochondrial oxygen consumption using a Clark electrode. Methods Mol Biol 837: 63-72.
    4. Plitzko, B., Kaweesa, E. N. and Loesgen, S. (2017). The natural product mensacarcin induces mitochondrial toxicity and apoptosis in melanoma cells. J Biol Chem 292(51): 21102-21116.
    5. Serill, J. D., Tan, M., Fotso, S., Sikorska, J., Kasanah, N., Hau, A. M., McPhail, K. L., Santosa, D. A., Zabriskie, T. M., Mahmud, T., Viollet, B., Proteau, P. J. and Ishmael, J. E. (2015). Apoptolodins A and C activate AMPK in metabolically sensitive cell types and are mechanistically distinct from oligomycin A. Biochem Pharmacol 93(3): 251-256.
    6. TeSlaa, T. and Teitell, M. A. (2014). Techniques to monitor glycolysis. Methods Enzymol 542: 91-114.
    7. Wu, M., Neilson, A., Swift, A. L., Moran, R., Tamagnine, J., Parslow, D., Armistead, S., Lemire, K., Orrell, J., Teich, J., Chomicz, S. and Ferrick, D. A. (2007). Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells. Am J Physiol Cell Physiol 292(1): C125-136.
    8. Zheng, J. (2012). Energy metabolism of cancer: Glycolysis versus oxidative phosphorylation (Review). Oncol Lett 4(6): 1151-1157.

简介

哺乳动物细胞通过线粒体（氧化磷酸化）和非线粒体（糖酵解）代谢产生ATP。已知癌细胞使用不同的策略重新编程它们的代谢以满足能量和合成代谢需要（Koppenol等人，2011; Zheng，2012）。此外，每个癌症组织都有其自己的个体代谢特征。线粒体不仅在能量代谢中起关键作用，而且在细胞的细胞周期调控中也起关键作用。因此，线粒体作为抗癌治疗的潜在靶标已经出现，因为它们在结构和功能上与其非癌对应物不同（D'Souza等人，2011）。我们详细介绍了利用海马XF24细胞外通量分析仪（图1）测量活细胞中氧耗率（OCR）和细胞外酸化率（ECAR）测量的方案。 Seahorse XF24细胞外通量分析仪持续测量细胞上清液中的氧浓度和质子流量（Wu等人，2007）。这些测量结果在OCR和ECAR值中转换，并能够直接定量线粒体呼吸和糖酵解。有了这个协议，我们试图评估三种不同癌细胞系的基线粒体功能和线粒体应激反应细胞毒性测试先导化合物甲磺卡西林，以研究其作用机制。将细胞铺在XF24细胞培养板中并保持24小时。在分析之前，将培养基替换为无缓冲的DMEM pH7.4，然后使细胞在非代谢通量分析前使用Seahorse XF在非CO 2孵育器中平衡以允许精确测量Milli-pH单位改变。 OCR和ECAR在基础条件下和通过药物注射端口注射化合物后进行测量。通过描述的方案，我们评估了三种细胞系的基本能量代谢谱以及响应于我们的测试化合物的线粒体功能的关键参数以及通过序贯添加线粒体扰动剂寡霉素，FCCP和鱼藤酮/抗霉素A.

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图1.海马实验概述

【背景】天然产物是从天然来源分离出来的小分子。在过去的一个世纪里，这些分子在治疗人类疾病，特别是灵感化疗药物方面起到了重要作用。代谢产物如紫杉醇，长春新碱和阿霉素已经彻底改变了我们如何治疗恶性肿瘤和其他天然产物，例如雷帕霉素，寡霉素和巴弗洛霉素，被用作分子探针，并能够在实验室中进行生物化学和细胞过程的分子研究。在研究细胞毒性天然产物mensacarcin的作用机制时，我们发现荧光标记的mensacarcin探针很大程度上定位于线粒体中（Plitzko等人，2017）。为了调查mensacarcin的细胞毒性性质是否可能来源于干扰线粒体功能，我们试图检查mensacarcin对细胞生物能量学的影响。使用海马细胞外通量分析仪，我们实时监测细胞氧耗率（OCR）和细胞外酸化率（ECAR），作为线粒体呼吸和糖酵解的测量值（Wu等人，2007; Serill 等人，2015）。海马XF24细胞外通量分析仪可以连续直接定量线粒体呼吸和活细胞的糖酵解。该仪器采用24孔板格式的传感器盒，每个传感器配备两个嵌入式荧光基团：一个通过氧气（O 2）淬灭，另一个对pH的变化敏感。在测量期间，传感器盒在细胞单层上方降低200μm，形成约2μl的微腔。海马仪器包含光纤束，可发射光线，激发荧光团，然后测量荧光团发射的变化。由瞬变微腔形成的非常小的测试体积可实时进行敏感，精确和无损的参数测量。自动计算氧气浓度和pH值的变化，并报告为氧气消耗率（OCR）和额外细胞酸化率（ECAR）。一旦完成测量，传感器就会升起，这样可以使上面的较大介质体积与瞬变微腔中的介质混合，将值恢复到基线。传感器盒含有端口，可在化验测量过程中每孔连续添加多达四种化合物。

利用所描述的方案，我们评估了三种细胞系（HCT-116，SK-Mel-28和SK-Mel-5）的能量代谢（图6）。发现mensacarcin的添加对黑素瘤细胞的基础OCR具有显着影响并且对ECAR没有增加的作用。通常观察到糖酵解的增加作为补偿反应。线粒体是细胞能量代谢所必需的，并且在凋亡细胞死亡中具有关键作用。线粒体呼吸作用的改变或促凋亡蛋白和抗凋亡蛋白之间的平衡可诱导线粒体失败。为了深入了解黑色素瘤细胞诱导的线粒体损伤，我们通过将细胞连续暴露于描述的线粒体扰动试剂来评估线粒体呼吸作用的关键参数。加入我们的测试化合物mensacarcin后，我们依次加入寡霉素，FCCP和最后的鱼藤酮和抗霉素A（图5）。寡霉素抑制ATP合酶并降低OCR，FCCP将氧消耗与ATP产生解偶联并将OCR提高至最大值，抗霉素A和鱼藤酮靶向电子传递链并将OCR降低至最小值。线粒体压力测试方案提供关于基底呼吸，与ATP相关的呼吸，质子泄漏，最大呼吸能力和细胞非线粒体呼吸的信息。因此，该测定可用于提供对直接靶向线粒体呼吸的化合物的作用机制的了解。

传统的线粒体功能或糖酵解测量需要氧电极，或使用比色或荧光检测的试剂盒和染料（Li和Graham，2012; TeSlaa和Teitell，2014）。这些方法大多是侵入性和繁琐的方法，只允许低的样品通量。相比之下，带有传感器盒系统的海马分析仪分析可以实时测量线粒体呼吸和糖酵解，并且无需任何染料或标签即可进行非侵入性检测。细胞能量代谢研究在哺乳动物细胞生物学的所有领域都是非常热门的话题。以下协议是为癌症生物学研究人员开发的，但适用于所有哺乳动物细胞系统能量代谢研究的方法。

关键字：生物能量学, 海马XF, 线粒体代谢, 糖酵解, 线粒体呼吸

材料和试剂

  1. CELLSTAR组织培养板96孔（Greiner Bio One International，目录号：655180）
  2. 无菌架式移液枪头（1毫升和200微升）（VWR，产品目录号：613-0738; 613-0742）
  3. 无菌盆（Corning，Costar ®，目录号：4870）
  4. 无菌试剂管（15和50毫升）（VWR，产品目录号：89039-668; 89039-662）
  5. 无菌血清移液管（5,10,25,50ml）（Fisher Scientific，目录号：13-678-11,13-678-11D，13-678-11E，13-678-11F）
  6. 玻璃瓶（500毫升）（Fisher Scientific，目录号：FB8001000）
  7. HCT-116，SK-Mel-5和SK-Mel-28细胞（ATCC，目录号：CCL-247，HTB-70，HTB-72）
  8. 海马XF24 FluxPak（包括传感器滤芯，组织培养板，校准溶液和校准板）（Agilent Technologies，Santa Clara，CA）
  9. 胰蛋白酶/ EDTA（0.25％/ 2.21mM）（Corning，目录号：25-053-C1）
  10. 1×Ca2 + / Mg2 + - 不含DPBS（Thermo Fisher Scientific，Gibco TM，目录号：14190250）
  11. 液体达尔伯克改良伊格尔培养基（DMEM）（Corning，目录号：10-013）
  12. 胎牛血清（FBS）（Atlanta Biologicals，目录号：S11150）

  13. 青霉素/链霉素溶液100倍（Corning，目录号：30-002-Cl）
  14. 不含HEPES（Corning，目录号：50-013）的不含Na 2 HCO 3的粉末Dulbecco改良的Eagle's培养基（DMEM）
  15. 氢氧化钠（NaOH）（VWR，目录号：97064-476）
  16. Oligomycin（Merck，目录号：495455-10MG）
  17. DMSO（VWR，目录号：BDH1115-1LP）
  18. FCCP（Cayman Chemical，目录号：15218）
  19. 鱼藤酮（Cayman Chemical，目录号：13995）
  20. 抗霉素A（Enzo Life Sciences，目录号：ALX-380-075-M005）
  21. 培养基（10％（v / v）FBS）（见食谱）
  22. 检测介质（见食谱）
  23. NaOH（1M）（见食谱）
  24. Oligomycin（10μM）（见食谱）
  25. FCCP（5μM）（见食谱）
  26. 鱼藤酮（5μM）/抗霉素A（5μM）（见食谱）

设备

  1. 血细胞计数器（Hausser Scientific，目录号：1490）
  2. 生物安全柜II类，A2型（NuAire，型号：NU-425-400ES）
  3. 海马XF细胞外通量分析仪（安捷伦科技公司，加利福尼亚州圣克拉拉）
  4. Pipet-Lite移液器XLS STD 20 XLS（Mettler Toledo，Rainin，型号：SL-2XLS +）
  5. Pipet-Lite移液器XLS STD 200（Mettler Toledo，Rainin，型号：SL-200XLS +）
  6. Pipet-Lite移液器XLS 1000（梅特勒托利多，Rainin，型号：SL-1000XLS +）
  7. 多通道Pipet-Lite移液器XLS 8-CH 1200（梅特勒托利多，Rainin，型号：L8-1200XLS +）
  8. 多道移液器移液管XLS 8-CH 200（梅特勒托利多，Rainin，型号：L8-200XLS +）
  9. 吸气泵
  10. 加湿的非CO 2培养箱（XF Prep Station; Agilent Technologies，Santa Clara，CA）
  11. 浅水浴（Thermo Fisher Scientific，Thermo Scientific TM，型号：Precision 180）
  12. 移液器控制器（BrandTech Scientific，型号：Accu-Jet Pro，目录号：26330）
  13. 加湿的，37℃，5％CO 2培养箱（Eppendorf，型号：Galaxy 170R）
  14. -20°C生物医学冷冻机（三洋，型号：MDF-U731M）
  15. 高压灭菌器（统一灭菌器系统，型号：SSR-3A，ADVPB）
  16. 倒置光学显微镜（尼康仪器，型号：Eclipse TS100）
  17. 具有半微电极（Thermo Fisher Scientific，Thermo Scientific TM，型号：Orion Star TM A211，具有ROSS 8103BN电极的Thermo Fisher pH计（Thermo Fisher Scientific，Thermo Scientific ，产品目录号：8103BN）

软件

  1. 海马生物科学XF24软件
  2. Excel（微软）
  3. GraphPad Prism 5.0（GraphPad Software，Inc.，La Jolla，CA）

程序

  1. 播种密度的优化
    在最初的实验中，每种细胞类型都需要最佳的接种密度。通常，细胞密度范围为每孔10,000至60,000个细胞，并且可以在细胞系中广泛变化。第一个取向点可以是约为约1的汇合点的单元格编号。在96孔细胞培养板中95％过夜，因为接种表面与海马培养板相当。在测定当天，播种数应该能够提供一个融合且健康且一致的单层。
    1. 使用两步接种技术将HCT-116，SK-Mel-5和SK-Mel-28细胞接种在Seahorse XF24细胞培养板中，浓度范围为10,000至30,000个细胞/孔，如以下程序B图2）。
      推荐三次重复种植细胞

      图2.用于细胞密度评估的平板布局这里显示了SK-Mel-5和SK-Mel-28细胞系的示例性播种布局（对于HCT-116细胞的接种密度在第二个板;未显示）。

    2. 然后按照程序E（无需将化合物装入端口）在XF24仪器中使用表1命令分析细胞。

      表1.用于细胞密度评估的协议命令


      如图3所示，在所有三种细胞系中都观察到随着细胞密度增加OCR值线性增加。对于SK-Mel-28和SK-Mel-5，ECAR值开始以20,000个细胞/孔平稳下降，而对于HCT-116而言，ECAR值以低得多且稳定增加。因此，选择SK-Mel-28和SK-Mel-5的20,000个细胞/孔的种子数目和HCT-116的35,000个细胞/孔的接种数目以确保在线性响应范围内，同时具有高读数值以观察增加以及OCR和ECAR的减少。


      图3.测定条件的优化：根据三种不同细胞系的接种密度评估OCR和ECAR

  2. 将细胞接种到Seahorse XF24组织培养板（第1天）
    注意：使用良好的无菌细胞培养技术进行细胞接种和生长。使用两步接种方法获得一致的均匀单层，这对获得一致和准确的数据至关重要：
    1. 预温培养基，胰蛋白酶溶液和DPBS至37°C。
    2. 对于贴壁细胞，用DPBS洗涤细胞，并添加胰蛋白酶，并等待细胞开始分离。添加含血清的培养基使胰蛋白酶失活并上下移液以制造均匀的细胞悬液。使用血细胞计数器计数细胞，并将细胞重悬于生长培养基中至所需的最终浓度，以100μl种子。
    3. 将100μl细胞悬液放入Seahorse XF24组织培养板中。
      仅在背景校正孔（A1，B4，C3，D6）中放置介质（无细胞）。
    4. 让培养板在生物罩中放置1小时而不移动它（为了让细胞均匀地沉降）。
    5. 将培养板置于培养箱（37℃，5％CO 2）中4小时。
    6. 小心添加150μL生长培养基（最终体积250μL）。以一定角度握住移液器尖端并将其添加到井侧，以不破坏新连接的细胞层。
    7. 让细胞在37℃，5％CO 2下过夜生长。

    注意：以下步骤在无菌技术的情况下执行，但请注意尽可能保持电池和设备的清洁。＆nbsp;

  3. 水合物传感器（第1天）
    1. 打开XF 24 FluxPak并取出传感器盒（绿色）和校准板（清除）（图4）。


      图4.海马XF 24传感器盒。 A.传感器盒位于校准板顶部并显示注射端口。 B.传感器板的底部显示嵌有荧光团的传感器。

    2. 将传感器盒（传感器向上）放在校准板旁边（小心不要接触传感器）。

    3. 用1毫升Seahorse XF校准剂填充校准板的每个孔。
    4. 将传感器盒放到校准板上，将传感器浸入校准液中（小心不要用传感器接触墙壁）。
    5. 置于非CO 2 37℃培养箱中过夜。为了防止XF Caldence的蒸发，请确认孵化器是否适当加湿。

  4. 仪器的稳定（第1天）
    1. 打开XF24分析仪，打开Seahorse Bioscience软件并登录。
    2. 写测定模板。在规划和编写化验协议时，请小心不要创建比无缓冲介质中的细胞可以在没有CO 2的情况下管理更长的协议。取决于细胞类型，这是2-3小时。如果有疑问，可以在海马测定后进行细胞活力测定。
    3. 保持XF24分析仪在XF24软件运行过夜并登录，以确保平衡至37°C。

  5. 海马测定（第2天）
    1. 检查细胞汇合。需要均匀的细胞间隔，没有大的细胞团或空白斑块，因为这可能会影响数据的准确性。
    2. 预温分析培养基至37°C。
    3. 预热化合物，必要时用NaOH（1M）调节至pH 7.4。
    4. 在Seahorse XF24组织培养板中进行培养基交换：
      1. 使用多通道移液器移除150μl生长培养基。

      2. 使用多通道移液器加入1 ml检测介质

      3. 用多通道移液器取出1毫升

      4. 用多通道移液器（575μl终体积）添加475μl检测介质
      5. 将细胞培养板置于CO 2自由培养箱中培养约2小时。 60分钟。
    5. 加载所需化合物的墨盒：
      1. 预热化合物至37°C。
      2. 将50-100μl化合物装入适当的药筒（用于线粒体压力测试：64μl进入A端口，71μlB端口，79μlC端口，88μlD端口）。 （见注1）将等量的测定培养基装入背景孔的等效端口（见注2）。
      3. 放回培养箱（非CO 2）中10分钟以再次加热至37℃。小心操作，只能握住校准板进行搬运。尽可能少移动。
    6. 校准和运行海马测定：
      1. 在Seahorse XF24软件中加载分析模板。
      2. 按绿色的“开始”按钮。
      3. 确保加载正确的协议，正确的保存目录并保存名称。
      4. 按'开始'。
      5. 将带有校准板的传感器盒装入仪器托盘（凹槽位于前方，左侧角落，确保平板在所有8个标签之间正确平放）
      6. 按照屏幕上的说明进行校准和平衡传感器。
      7. 一旦完成平衡步骤，取出校准板并更换为细胞培养板。

  6. 协议命令（线粒体压力测试，表2，图5）

    表2.线粒体压力测试的协议命令

数据分析

结果最初使用海马XF数据查看器进行审查，该数据查看器将数据自动保存为MS Excel（.xls）文件。图形和统计分析使用GraphPad Prism进行。通过非参数Kruskal-Wallis检验以及随后的Dunn's多重比较事后检验来评估观察到的细胞系生物能量学差异的显着性。在所有情况下， P ＆lt; 0.05被认为是显着的。实验值以平均值±标准偏差（图5）或箱形图（图6）报告。


图5.线粒体压力测试在不同浓度注射甲磺卡西林（黑色箭头）后，测量SK-Mel-28细胞的OCR，然后连续注射寡霉素（1μM），FCCP（0.5μM ）和抗霉素A（0.5μM）/鱼藤酮（0.5μM）（n = 3）。


图6. SK-Mel-28，SK-Mel-5和HCT-116细胞的基础生物能状态通过分析OCR / ECAR比率评估每个细胞系的基础能量代谢。使用与上述相同的方案获得OCR和ECAR，但没有注射化合物。协议命令由8个测量的一个循环组成。进行了几次单独的测定（n = 25）。

笔记

  1. 以角度吸入端口，不要碰到底部，不要轻敲以防止泄漏。液体只能被毛细管力量保存。
  2. 使用含有与化合物相同浓度的DMSO的化验培养基加载背景孔的端口是强制性的，以说明任何DMSO对细胞的影响。
  3. 一旦注入孔中，化合物以1:10稀释。这将在细胞培养孔中分别得到1μM寡霉素和0.5μMFCCP，鱼藤酮和抗霉素A的终浓度。

食谱

  1. 培养基（10％（v / v）FBS）
    注意：在无菌条件下在层流罩中工作
    1. 开放式液体DMEM瓶
    2. 用无菌血清吸管取出55毫升，并丢弃液体

    3. 用无菌血清移液管加50 ml FBS
    4. 加入5毫升青霉素/链霉素溶液
    5. 在4°C储存
  2. 测定培养基（无菌，无缓冲，250毫升）
    注意：在无菌条件下在层流罩中工作

    1. 高压灭菌器250毫升超纯H 2 O在玻璃瓶中
    2. 将不含NaHCO 3且不含HEPES的3,34g粉末DMEM溶解在250ml高压灭菌的H 2 O中。
    3. 温暖至37°C
    4. 用NaOH（1M）调节至pH 7.40
    5. 在4°C储存
  3. NaOH（1M）
    将4g NaOH丸粒溶解在100ml高压灭菌的H 2 O中
  4. 寡霉素（10μM）
    1. 在海马试验当天新鲜准备（第2天）（见注1）
    2. 在1ml DMSO中制备1mM溶液：将0.7911mg寡霉素溶解在DMSO中
    3. 在测定培养基（1％DMSO）中稀释至10μM：将20μl1 mM寡霉素吸入1,980μl测定培养基中
    4. 温至37°C，如有必要，用NaOH（1M）调节至pH 7.4。
  5. FCCP（5μM）
    1. 在海马试验当天新鲜准备（第2天）（见注1）
    2. 在DMSO中制备50mM溶液：将2.54mg FCCP溶解在200μlDMSO中
    3. 稀释至500μM：将10μl50 mM FCCP吸入990μlDMSO中。
    4. 在测定培养基（1％DMSO）中稀释至5μM：将20μl500μMFCCP吸入1,980μl测定培养基中
    5. 温至37°C，必要时用NaOH（1M）调节至pH 7.4。
  6. 鱼藤酮（5μM）/抗霉素A（5μM）

    1. 在海马测定当天新鲜准备（第2天）（见注1）。
    2. 准备在DMSO中的50mM溶液：在200μlDMSO中溶解3.94mg鱼藤酮和5.49mg抗霉素A
    3. 稀释至1mM：将10μl50mM鱼藤酮/抗霉素A吸入490μlDMSO中
    4. 在测定培养基（0.5％DMSO）中稀释至5μM：将20μl1 mM鱼藤酮/抗霉素A吸入1,980μl测定培养基中
    5. 温热至37°C，如有必要，用NaOH（1M）调节至pH 7.4。

致谢

这项工作主要由俄勒冈州立大学启动资金支持。 B.P.感谢DFG（Deutsche Forschungsgemeinschaft）博士后资助（PL 799 / 1-1）。我们希望感谢Napur Pande博士提供SK-Mel-28细胞，并感谢Bioviotica（Prof. Axel Zeeck博士和Hans-Peter Kroll）提供的mensacarcin。我们要感谢Elizabeth N. Kaweesa对本协议的帮助，并感谢Jeffrey D. Serrill博士和Jane E. Ishmael教授提供关于海马实验的信息和反馈。作者声明不存在利益冲突或利益冲突。

参考

  1. D'Souza，G.G.，Wagle，M.A。，Saxena，V。和Shah，A。（2011）。 在癌症治疗中靶向线粒体的方法 Biochim Biophys Acta > 1807（6）：689-696。
  2. Koppenol，W.H.，Bounds，P.L。和Dang，C.V。（2011）。 Otto Warburg对当前癌症代谢概念的贡献 Nat Rev Cancer 11（5）：325-337。
  3. Li，Z。和Graham，B.H。（2012）。 使用Clark电极测量线粒体耗氧量方法Mol Biol 837：63-72。
  4. Plitzko，B.，Kaweesa，E.N.和Loesgen，S。（2017）。 天然产物mensacarcin诱导黑色素瘤细胞的线粒体毒性和细胞凋亡。 J Biol Chem 292（51）：21102-21116。
  5. Serill，JD，Tan，M.，Fotso，S.，Sikorska，J.，Kasanah，N.，Hau，AM，McPhail，KL，Santosa，DA，Zabriskie，TM，Mahmud，T.，Viollet， Proteau，PJ和Ishmael，JE（2015）。 细胞凋亡素A和C在代谢敏感细胞类型中激活AMPK，并且与寡霉素A在机理上不同。 （Biochem Pharmacol）93（3）：251-256。
  6. TeSlaa，T.和Teitell，M.A。（2014）。 监测糖酵解的技术 Methods Enzymol 542：91 -114。
  7. Wu，M.，Neilson，A.，Swift，AL，Moran，R.，Tamagnine，J.，Parslow，D.，Armistead，S.，Lemire，K.，Orrell，J.，Teich，J.，Chomicz ，S.和Ferrick，DA（2007）。 多参数代谢分析揭示了减弱的线粒体生物能功能与人类肿瘤细胞中增强的糖酵解依赖性之间的密切联系。 Am J Physiol Cell Physiol 292（1）：C125-136。
  8. 郑，J。（2012）。 癌症的能量代谢：糖酵解与氧化磷酸化（综述） Lett 4（6）：1151-1157。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. 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. DOI: 10.21769/BioProtoc.2850.
  2. Plitzko, B., Kaweesa, E. N. and Loesgen, S. (2017). The natural product mensacarcin induces mitochondrial toxicity and apoptosis in melanoma cells. J Biol Chem 292(51): 21102-21116.
分类
癌症生物学 > 细胞能量学 > 细胞生物学试验 > 新陈代谢
细胞生物学 > 细胞新陈代谢 > 其它化合物
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