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Assessment of Brown Adipocyte Thermogenic Function by High-throughput Respirometry
高通量呼吸运动计量法测量棕色脂肪细胞的产热功能评估   

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Abstract

Brown adipose tissue (BAT) has the unique ability to dramatically increase mitochondrial uncoupled fuel oxidation for thermogenesis in response to adrenergic stimulation. A key parameter in assessing brown adipocyte thermogenic capacity is mitochondrial uncoupling as determined by respiration. Measuring mitochondrial oxygen consumption rate (OCR) therefore provides valuable information to study the regulation and dysregulation of fuel metabolism and energy expenditure. Adding measurements of mitochondrial membrane potential allows for more in-depth interpretation of the respirometry data. Here we provide protocols for measuring respiration in adherent intact and plasma membrane permeabilized brown adipocytes using the Seahorse XF Analyzer. In the protocol Part I, a combination of norepinephrine and free fatty acids are used to induce uncoupled respiration. The ATP Synthase inhibitor oligomycin, the chemical uncoupler FCCP, and the complex III inhibitor Antimycin A are then used to measure coupled, maximal, and non-mitochondrial oxygen consumption, respectively. In the protocol Part II, the plasma membrane is permeabilized with recombinant perfringolysin O, a cholesterol-dependent cytolysin that oligomerizes into pores exclusively in the plasma membrane. This permits experimental control of metabolite availability without separating mitochondria from the native cell environment.

Part I. Intact brown adipocyte respiration

Materials and Reagents

Note: Rotenone, antimycin, oligomycin, and FCCP are toxic and light sensitive. Wear personal protective equipment when handling and store stocks in the dark.

  1. 3-4 weeks-old C57BL6/J mice
    Note: Isolate primary pre adipocytes from brown adipose tissue, then differentiate them in an XF-24 well cell culture microplate for 7 days.
  2. XF assay 24‐well cartridge and cell culture microplate (Seahorse Bioscience, catalog numbers: 100850-001 and 100777-004 )
  3. XF assay 24-well cartridge and utility plate
  4. DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 12100-046 )
  5. ewborn calf serum (Sigma-Aldrich, catalog number: N4887 )
  6. HEPES (Corning, catalog number: 25-060-Cl )
  7. L-Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030-164 )
  8. Penicillin-Streptomycin Solution (100x) (Corning, catalog number: 30-002-Cl )
  9. Sodium L-ascorbate (Sigma-Aldrich, catalog number: A4034 )
  10.  Rosiglitazone (used as differentiation agent) (Toronto Research Chemicals, catalog number: R693500 )
  11. Insulin from Porcine Pancreas (Sigma-Aldrich, catalog number: I5523 )
  12. XF-assay/base medium (Seahorse Bioscience, Catalog number: 102365-100 )
  13. D-(+)-Glucose (Sigma-Aldrich, catalog numbers: G8270 )
  14. XF Calibrant solution (Seahorse Bioscience, Catalog number: 100840‐000 )
  15. L-(-)Norepinephrine(+)-bitartrate salt monohydrate (Sigma-Aldrich, Catalog number: N5785 )
  16. Free fatty acids (Palmitic acid or Oleate) (Sigma-Aldrich, catalog number: P0500 )
  17. DMSO (Sigma-Aldrich, catalog number: D2650 )
  18. RPMI medium (no glucose) (Thermo Fisher Scientific, GibcoTM, catalog number: 11879-020 )
  19. Fatty‐acid‐free BSA (CalBiochem, catalog number: 126575 )
  20. BAT differentiation media (see Recipes)
  21. Seahorse assay media (SH assay media) (see Recipes)
  22. Free fatty acid conjugated to the BSA (see Recipes)
  23. Norepinephrine (see Recipes)
  24. Oligomycin (Sigma-Aldrich, catalog number: 75371 ) (see Recipes)
  25. Antimycin A from Streptomyces sp. (Sigma-Aldrich, catalog number: A8674 ) (see Recipes)
  26. Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (Sigma-Aldrich, catalog number: C2920 ) (see Recipes)
  27. Sodium pyruvate (Sigma-Aldrich, catalog number: P5280 ) (see Recipes)

Equipment

  1. CO2-free incubator
  2. 8% CO2 incubator
  3.  XF24 extracellular flux analyzer (Seahorse Bioscience)
  4. Cell culture hood
  5. pH meter
  6. 37 °C Water bath

Procedure

  1. Isolate preadipocytes from 3-4 weeks-old C57BL6/J mice according to Cannon and Nedergaard (2001). Resuspend cell pellet in 4 ml of BAT differentiation medium after BAT collagenase digestion and washing steps.
  2. Seed 70 μl of resuspended pellet into each well of a V7 XF 24-well cell culture plate, allow cells to attach for 1 h in cell culture hood, and then add 180 μl BAT differentiation medium and put in 8% CO2 incubator (getting an exact cell count is difficult at this stage because of the debris and red blood cells left over from isolation procedure). The next day, gently wash the cells twice with 150 μl medium to make sure that the red blood cells are washed out and add 250 μl of fresh medium. Replace the medium every other day for the next 7 days or until the preadipocytes differentiate. Differentiation can be visualized by lipid droplet formation in adipocytes or assessed by Western blot analysis of UCP1 expression (see Representative data Figures 1 and 2).
  3. Before beginning the respiratory assay, soak the cartridge with 1 ml of XF Calibrant per well for at least 3 h in CO2-free incubator.
  4. Wash cells once with SH assay media and then add 450 μl of SH assay medium to each of the 24 wells in the plate.
  5. Incubate plate for 30 min at 37 °C in CO2-free incubator before loading into the XF 24 extracellular analyzer.
  6. During these 30 min, load the ports of the cartridge containing the oxygen probes with the compounds to be injected during the assay (50 μl/portA, 55 μl/portB, 60 μl/portC, 65 μl/portD) and calibrate the cartridge.
  7. Run seahorse assay with 2 min mix and 4 min measurement (no wait). 6 measurements under basal, 5 measurements after Oligomycin and FCCP injection and more than 5 measurements after Antimycin A injection are recommended.

Representative data


Figure 1. Brown adipocyte differentiation with insulin and rosiglitazone.
a) UCP1 immunostaining of differentiated (with rosiglitazone) vs. undifferentiated cells (no rosiglitazone). Note the difference in fluorescence intensity. b) Western blot for UCP1and porin in differentiated brown adipocytes in the presence and absence of insulin and rosiglitazone. (Wikstrom et al., 2014)


Figure 2. Representative images of pre-adipocytes 24 h after isolation and mature brown-adipocytes after 7 days of differentiation



Figure 3. Representative OCR plot with injection of A) NE+P, NE, or palmitate alone, B) Oligomycin, and C) Antimycin A

Notes

  1. Successful NE activation of brown adipocytes expressing UCP1 results in a rapid increase in oxygen consumption rate. If OCR increase is <3-fold this suggests the brown adipocytes have not been sufficiently differentiated.

Recipes

  1. BAT differentiation media
    Prepare stock (good for 6 weeks at 4 °C) of DMEM supplemented with:
    10% newborn calf serum
    10 mM HEPES
    4 mM glutamine
    50 IU of penicillin
    50 μg streptomycin
    100 μg/ml sodium L-ascorbate
    Prepare 50 ml of BAT differentiation media (good for 1 week at 4 °C):
    50 ml of Stock
    60 nM insulin
    1 μM rosiglitazone
  2. Seahorse assay media (SH assay media) (PH=7.4 and filtered)
    XF-assay medium
    Stock contains:
    0.8 mM Mg2+
    1.8 mM Ca2+
    143 mM NaCl
    5.4 mM KCl
    0.91 mM NaH2PO4
    2 mM L-Ala-Gln (Glutamax)
    Phenol red 3 mg/ml
    On day of experiment: Add 3 mM glucose freshly to XF-assay medium, pH to 7.4 at 37 °C and sterile filter
  3. Free fatty acid conjugated to the BSA (0.4 mM; FFA: BSA, 4:1)
    Dissolve free fatty acids (Palmitate or Oleate) in DMSO to a final concentration of 0.4 M and then dissolve this solution at 43 °C in RPMI medium (No Glucose) containing 6.7% fatty‐acid‐free BSA to make a 10x stock (4 mM)
    Aliquot and store in -80 °C
  4. Norepinephrine
    Freshly prepare L-(-)Norepinephrine(+)-bitartrate salt monohydrate in SH assay media at stock concentrations of 10 mM and use at final concentrations of 1 μM
  5. Oligomycin
    Prepare stock of 20 mM in DMSO and store in -20 °C
    On day of experiment, dilute in SH assay media to prepare 50 μM (10x) solution to be loaded into cartridge ports
    Final concentration will be 5 μM
  6. Antimycin A
    Prepare stock of 40 mM in DMSO and store in -20 °C
    On day of experiment, dilute in SH assay media to prepare 80 μM (10x) solution to be loaded into cartridge ports
    Final concentration will be 8 μM
  7. Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP)
    Prepare stock of 20 mM in DMSO and store in -20 °C
    On day of experiment, dilute in SH assay media containing 100 mM Na pyruvate to prepare 50 μM (10x) solution to be loaded into cartridge ports
    Final concentration will be 5 μM with 10mM sodium pyruvate
  8. Sodium pyruvate
    Dissolve in SH assay media at the concentration of 100 mM (pH=7.4 and filtered)
    Final concentration is 10 mM
    Substrate
    Solvent
    Concentration (10x)/Media
    Final concentration
    Comment
    Norepinephrine
    -
    10 μM/SH assay media
    1 μM

    FFA: BSA
    RPMI no glucose
    4 mM/RPMI no glucose
    0.4 mM; 4:1

    Oligomycin
    DMSO
    50 μM/SH assay media
    5 μM

    Antimycin A
    DMSO
    80 μM/SH assay media
    8 μM

    FCCP
    DMSO
    50 μM/SH+ Na Pyruvate
    5 μM

    Na pyruvate
    -
    100 mM/SH assay media
    10 mM
    pH=7.4 and filter
     

Part II. Permeabilized brown adipocyte respiration

Materials and Reagents

  1. D-Mannitol (Sigma-Aldrich, catalog number: M9647 )
  2. Sucrose (Thermo Fisher Scientific, catalog number: S5 )
  3. Potassium Phosphate Monobasic (KH2PO4) (Thermo Fisher Scientific, catalog number: P285 )
  4. Magnesium Chloride Hexahydrate (MgCl2) (Thermo Fisher Scientific, catalog number: M33 )
  5. HEPES (Sigma-Aldrich, catalog number: H4034 )
  6. Ethylene glycol-bis(β-aminoethyl ether)-N,N,N,N'-tetraacetic acid tetrasodium salt (EGTA) (Sigma-Aldrich, catalog number: E8145 )
  7. Fatty-Acid Free BSA (EMD Millipore Corporation, CalBiochem, catalog number: 126575 )
  8. L-(−)-Malic acid (Sigma-Aldrich, catalog number: 02288 )
  9. Succinate (Sigma-Aldrich, catalog number: S3674 )
  10. Oligomycin A (Sigma-Aldrich, catalog number: 75371 )
  11. Antimycin A from Streptomyces sp. (Sigma-Aldrich, catalog number: A8674 )
  12. Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (Sigma-Aldrich, catalog number: C2920 )
  13. Rotenone (Sigma-Aldrich, catalog number: R8875 )
  14. (+)-Etomoxir sodium salt hydrate (Sigma-Aldrich, catalog number: E1905 )
  15. Adenosine 5’-diphosphate monopotassium salt dihydrate (Sigma-Aldrich, catalog number: A5285 )
  16. L-(-)-Carnitine (Thermo Fisher Scientific, Acros Organics, catalog number: 241040100 )
  17. Succinate (Sigma-Aldrich, catalog number: S3674 )
  18. L-(−)-Malic acid (Sigma-Aldrich, catalog number: 02288 )
  19. Sodium pyruvate (Sigma-Aldrich, catalog number: P5280 )
  20. Palmitoyl-L-carnitine chloride (Sigma-Aldrich, catalog number: P1645 )
  21. Palmitoyl coenzyme A lithium salt (Sigma-Aldrich, catalog number: P9716 )
  22. 3x MAS buffer (see Recipes)
  23. XF PMP reagent (Seahorse Bioscience, catalog number: 102504-100) (see Recipes)

Equipment

  1. Same equipment as intact brown adipocyte respiration protocol

Procedure

  1. Prepare cells as described in steps 1-2 of intact brown adipocyte respiration protocol.
  2. On the day of experiment, thaw 1x MAS and pre-made substrate solutions described in Recipes section 1.
  3. Prepare 10x solutions of inhibitors in 1x MAS. If using fatty acid substrates, prepare them freshly as described in Recipes section 2b.
    1. Soak the cartridge for at least 3 h in CO2-free incubator before loading.
    2. Load ports: 50 μl in Port A, 55 μl in Port B, 60 μl in Port C, 65 μl in Port D and calibrate cartridge.
    3. Prepare 7.5 nM PMP in 1x MAS in a volume sufficient for 450 μl per well.
    4. Gently wash cells with 1x MAS buffer one time.
    5. Completely evacuate wash and add 450 μl 1x MAS with 7.5 nM PMP. Incubation in CO2-free incubator is not necessary.
    6.  Immediately load the plate and run seahorse assay with 3 min mix and 2 min measurement (no wait). Make 2-3 measurements per condition. Do not exceed 1 h of assay time preceding Antimycin A injection as mitochondrial integrity will be compromised.

Representative data


Figure 4. Representative OCR plot of permeabilized BAT injected with A) Succinate+ADP+Rotenone, B) Oligomycin, and C) Antimycin

Notes

  1. The simplest way to ensure permeabilization is to detect respiration elicited by the membrane-impermeable substrate succinate. Succinate may also be used to titrate the optimal XF PMP reagent concentration
  2. Always load all four injections ports. If one port is not used, load with 1x MAS buffer.
  3. Use at least three technical replicates per condition.
  4. To ensure the quality of the data, make sure that pH is stable throughout the run and that oxygen tension returns to base level after every measurement.
  5. If OCR exceeds 2,000 pmol/min, go to ‘Special Operations’ tab in the seahorse excel data viewer, click on ‘Change O2 calc method’ and select the ‘Gain (Fixed)’ algorithm as the AKOS algorithm will not process high OCR data correctly. Always express Gain (Fixed) data as percent of baseline measurement.
  6. For more information on permeabilization see Divakaruni et al. (2014) and Salabei et al. (2014).

Recipes

  1. 3x MAS buffer
    Reagent
    Concentration
    Amount 1 L MAS
    Mannitol
    660 mM
    120.23 g
    Sucrose
    210 mM
    71.88 g
    KH2PO4
    30 mM
    4.08 g
    MgCl2
    15 mM
    15 ml of 1.0 M solution
    HEPES
    6 mM
    6 ml of 1.0 M solution
    EGTA
    3 mM
    12 ml of 0.25 M solution
    Fatty-Acid Free BSA
    0.6% (w/v)
    6.0 g
    Dissolve reagents in double-distilled water, pH to 7.2 with KOH, sterile filter, and store in -20 °C
    Optional: Dilute to 1X in double-distilled water, pH to 7.2 with KOH, sterile filter, and store in -20 °C
  2. XF PMP reagent
    Stock solution: 10 μM
    Storage: -20 °C
    Stock solutions
    Reagent
    MW
    Concentration
    Solvent
    Comments
    Malate
    134.09  
    1.0 M
    ddH2O   
    pH to 7.2 with KOH. Solution is strongly acidic, calculate molarity according to final volume.
    Succinate
    270.1
    1.0 M
    ddH2O
    pH to 7.2 with KOH
    Oligomycin
    791.06
    20 mM
    DMSO
    Complex V inhibitor
    Antimycin A
    548.63
    40 mM
    DMSO
    Complex III inhibitor
    FCCP
    254.17
    40 mM
    DMSO
    Chemical uncoupler
    Rotenone
    394.4
    40 mM
    DMSO
    Complex I inhibitor
    Etomoxir
    338.76
    40 mM
    DMSO
    CPT-1 inhibitor, blocks conversion of palmitoyl-coA to palmitoyl-carnitine
    ADP, K+ salt
    501.32
    Use as powder


    L-(-)-Carnitine   
    161.2
    Use as powder



    Substrates
    Substrate
    MW
    10x Conc. for port injection
    Additives (10x conc. for port injection)
    Succinate
    270.1
    100 mM
    40 mM ADP, 50 μM Rotenone
    Malate
    134.09
    30 mM
    40 mM ADP
    Pyruvate
    110.94
    100 mM
    40 mM ADP, 30 mM malate
    Palmitoyl-carnitine
    436.07
    1 mM
    40 mM ADP, 30 mM malate, 5 mM carnitine
    Palmitoyl-CoA
     1,004.94
    1 mM
    40 mM ADP, 30 mM malate, 5 mM carnitine

    1. Prepare succinate, malate, and pyruvate in 1x MAS buffer and pH with KOH to 7.2 prior to day of experiment. Sterile filter, aliquot, and store in -20 °C.
      1. For example, for a 5 ml solution of succinate, add 0.5 ml 1.0 M succinate, 100.2 mg ADP, 1.67 ml 3x MAS, and 2.83 ml ddH2O and adjust pH to 7.2 with KOH. (Add rotenone on day of experiment)
      2. For every 100 μl of additional volume as a result of pH titration, add 50 μl 3x MAS to maintain osmotic strength.
      3. For example, if final volume of the 5 ml succinate solution is 5.1 ml, add 50 μl 3x MAS. This will result in a slight dilution of reagents that should not significantly affect the assay. However, given this variability, we advise preparing a large batch of solution and use the same stocks for all experiments within a given project.
    2. For fatty acids, prepare solutions of additives (except carnitine) in 1x MAS buffer and pH with KOH to 7.2 prior to day of the experiment. Sterile filter, aliquot, and store in -20 °C. Add carnitine and fatty acid to solutions on the day of the experiment.

Acknowledgments

Permeabilized brown adipocyte respiration protocol adapted from Divakaruni et al. (2014). This work was supported by National Institutes of Health grants R 01 DK074778, R01 DK56690-11 and by the Swedish Research Council as well as DIABAT FP7, collaborative EU Grant HEALTH-F2-2011-278373 and the Wallenberg Foundation.

References

  1. Cannon, B. and Nedergaard, J. (2001). Cultures of adipose precursor cells from brown adipose tissue and of clonal brown-adipocyte-like cell lines. Methods Mol Biol 155: 213-224.
  2. Divakaruni, A. S., Rogers, G. W. and Murphy, A. N. (2014). Measuring mitochondrial function in permeabilized cells using the seahorse XF analyzer or a clark-type oxygen electrode. Curr Protoc Toxicol 60: 25 22 21-25 22 16.
  3. Salabei, J. K., Gibb, A. A. and Hill, B. G. (2014). Comprehensive measurement of respiratory activity in permeabilized cells using extracellular flux analysis. Nat Protoc 9(2): 421-438.
  4. Wikstrom, J. D., Mahdaviani, K., Liesa, M., Sereda, S. B., Si, Y., Las, G., Twig, G., Petrovic, N., Zingaretti, C. and Graham, A. (2014). Hormone‐induced mitochondrial fission is utilized by brown adipocytes as an amplification pathway for energy expenditure. EMBO J 33(5): 418-436.

简介

棕色脂肪组织(BAT)具有显着增加线粒体解偶联燃料氧化以响应肾上腺素刺激的热生成的独特能力。评估棕色脂肪细胞产热能力的关键参数是通过呼吸确定的线粒体解偶联。因此,测量线粒体氧消耗率(OCR)为研究燃料代谢和能量消耗的调节和失调提供了有价值的信息。添加线粒体膜电位的测量允许更加深入地解释呼吸数据。在这里我们提供使用Seahorse XF分析仪测量贴壁完整和质膜透性棕色脂肪细胞呼吸的协议。在方案部分I中,去甲肾上腺素和游离脂肪酸的组合用于诱导解偶联呼吸。然后使用ATP合酶抑制剂寡霉素,化学去偶联剂FCCP和复合物III抑制剂抗霉素A分别测量偶联的,最大的和非线粒体的氧消耗。在方案第II部分中,质膜用重组perfringolysin O透化,胆固醇依赖性细胞溶解素寡聚化成在质膜中专门的孔。这允许代谢物可用性的实验性控制,而不从天然细胞环境中分离线粒体。

部分I.完整的棕色脂肪细胞呼吸

材料和试剂

注意:鱼藤酮,抗霉素,寡霉素和FCCP是有毒和光敏感的。在黑暗中处理和储存库存时,请穿戴个人防护装备。

  1. 3-4周龄的C57BL6/J小鼠
    注意:从棕色脂肪组织中分离初级前脂肪细胞,然后在XF-24孔细胞培养板中将其分化7天。
  2. XF测定24孔盒和细胞培养微板(Seahorse Bioscience,目录号:100850-001和100777-004)
  3. XF测定24孔筒和实用板
  4. DMEM(Thermo Fisher Scientific,GibcoTM,目录号:12100-046)
  5. ewborn小牛血清(Sigma-Aldrich,目录号:N4887)
  6. HEPES(Corning,目录号:25-060-Cl)
  7. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM ,目录号:25030-164)
  8. 青霉素 - 链霉素溶液(100x)(Corning,目录号:30-002-Cl)
  9. 钠抗坏血酸钠(Sigma-Aldrich,目录号:A4034)
  10.  罗格列酮(用作分化剂)(Toronto Research Chemicals,目录号:R693500)
  11. 来自猪胰岛素的胰岛素(Sigma-Aldrich,目录号:I5523)
  12. XF-测定/基础培养基(Seahorse Bioscience,目录号:102365-100)
  13. D - (+) - 葡萄糖(Sigma-Aldrich,目录号:G8270)
  14. XF校准溶液(Seahorse Bioscience,目录号:100840-000)
  15. L - ( - )去甲肾上腺素(+) - 酒石酸盐一水合物(Sigma-Aldrich,目录号:N5785)
  16. 游离脂肪酸(棕榈酸或油酸酯)(Sigma-Aldrich,目录号:P0500)
  17. DMSO(Sigma-Aldrich,目录号:D2650)
  18. RPMI培养基(无葡萄糖)(Thermo Fisher Scientific,Gibco TM ,目录号:11879-020)
  19. 无脂肪酸BSA(CalBiochem,目录号:126575)
  20. BAT分化培养基(参见配方)
  21. 海马测定培养基(SH测定培养基)(参见配方)
  22. 游离脂肪酸与BSA结合(参见配方)
  23. 去甲肾上腺素(见配方)
  24. 寡霉素(Sigma-Aldrich,目录号:75371)(参见Recipes)
  25. 来自链霉菌属(Sigma-Aldrich,目录号:A8674)(参见Recipes)
  26. 羰基氰4-(三氟甲氧基)苯腙(FCCP)(Sigma-Aldrich,目录号:C2920)(参见配方)
  27. 丙酮酸钠(Sigma-Aldrich,目录号:P5280)(参见Recipes)

设备

  1. CO 2无孵化器
  2. 8%CO 2培养箱
  3.   XF24胞外通量分析仪(Seahorse Bioscience)
  4. 细胞培养罩
  5. pH计
  6. 37°C水浴

程序

  1. 根据Cannon和Nedergaard(2001)分离来自3-4周龄C57BL6/J小鼠的前脂肪细胞。在BAT胶原酶消化和洗涤步骤后,将细胞沉淀重悬于4ml BAT分化培养基中
  2. 将70μl重悬浮的沉淀物重悬于V7XF 24孔细胞培养板的每个孔中,使细胞在细胞培养罩中附着1小时,然后加入180μlBAT分化培养基并放入8%CO 2 孵化器(在这个阶段,由于从分离程序留下的碎片和红细胞,获得精确的细胞计数是困难的)。第二天,用150微升培养基轻轻洗涤细胞两次,以确保红细胞被洗出,并添加250微升的新鲜培养基。每隔一天更换培养基,接下来的7天或直到前脂肪细胞分化。分化可以通过脂肪细胞中的脂滴形成或通过UCP1表达的Western印迹分析来评估(参见代表性数据图??1和2)。
  3. 在开始呼吸测定之前,在CO 2 sub-free培养箱中用1ml XF Calibrant每孔浸泡小室至少3小时。
  4. 用SH测定培养基洗涤细胞一次,然后向板的24个孔中的每个孔中加入450μlSH测定培养基
  5. 在装载到XF 24细胞外分析仪中之前,在37℃下在CO 2无培养箱中孵育板30分钟。
  6. 在这30分钟内,在测定期间(50μl/端口A,55μl/端口B,60μl/端口C,65μl/端口D)装载含有氧探针的柱的端口并校准柱体。
  7. 运行海马测定,2分钟混合和4分钟测量(无等待)。 6次测量基础,5次测量后寡聚霉素和FCCP注射和超过5次测量后建议使用抗霉素A。

代表数据


图1.具有胰岛素和罗格列酮的棕色脂肪细胞分化 a)分化的(与罗格列酮)与未分化细胞(无罗格列酮)的UCP1免疫染色。注意荧光强度的差异。 b)在存在和不存在胰岛素和罗格列酮的情况下,在分化的棕色脂肪细胞中对UCP1和孔蛋白的Western印迹。 (Wikstrom 等人,2014)


图2.分离后24小时的前脂肪细胞和分化7天后成熟棕色脂肪细胞的代表性图像


图3.注射A)NE + P,NE或单独的棕榈酸,B)寡霉素和C)抗霉素A的代表性OCR图。

笔记

  1. 成功的NE活化表达UCP1的棕色脂肪细胞导致氧消耗速率的快速增加。如果OCR增加≤3倍,这表明棕色脂肪细胞未被充分分化。

食谱

  1. BAT分化培养基
    准备补充有以下物质的DMEM(在4℃下有效6周): 10%新生小牛血清
    10 mM HEPES
    4mM谷氨酰胺 50 IU青霉素
    50μg链霉素 100μg/ml L-抗坏血酸钠 准备50ml的BAT分化培养基(4℃下1周有效):
    50毫升的股票
    60 nM胰岛素
    1μM罗格列酮
  2. 海藻测定培养基(SH测定培养基)(PH = 7.4并过滤)
    XF测定培养基
    库存包含:
    0.8mM Mg 2+ +
    1.8mM Ca 2+ 2 +
    143 mM NaCl 5.4 mM KCl
    0.91mM NaH 2 PO 4 4·h/v 2mM L-Ala-Gln(Glutamax)
    酚红3mg/ml
    实验当天:将3mM葡萄糖新鲜加入到XF-测定培养基中,在37℃和无菌过滤器下pH为7.4,
  3. 与BSA结合的游离脂肪酸(0.4mM; FFA:BSA,4:1)
    将游离脂肪酸(棕榈酸盐或油酸盐)溶于DMSO至0.4M的最终浓度,然后在43℃下在含有6.7%无脂肪酸的BSA的RPMI培养基(无葡萄糖)中溶解该溶液,以制备10×储备液mM)
    等分并存储在-80°C
  4. 去甲肾上腺素
    在SH测定培养基中以10mM的原液浓度新制备L - ( - )去甲肾上腺素(+) - 酒石酸氢盐一水合物,并以1μM的终浓度使用
  5. 寡霉素
    准备20mM在DMSO中的储备液,并储存在-20℃下 在实验当天,稀释在SH测定培养基中以制备50μM(10x)溶液装载到盒端口中。
    最终浓度为5μM
  6. 抗霉素A
    准备40mM在DMSO中的储备液,并储存在-20℃下 在实验当天,在SH测定培养基中稀释以制备待装载到盒端口中的80μM(10x)溶液。
    最终浓度为8μM
  7. 羰基氰4-(三氟甲氧基)苯腙(FCCP)
    准备20mM在DMSO中的储备液,并储存在-20℃下 在实验当天,在含有100mM丙酮酸钠的SH测定培养基中稀释,以制备待装载到盒端口中的50μM(10x)溶液。
    最终浓度为5μM,用10mM丙酮酸钠
  8. 丙酮酸钠
    溶于SH试验介质中,浓度为100mM(pH = 7.4,过滤) 最终浓度为10mM。
    基材
    溶剂
    集中(10x)/媒体
    最终集中
    评论
    去甲肾上腺素
    -
    10μM/SH测定培养基
    1μM

    FFA:BSA
    RPMI无葡萄糖
    4mM/RPMI无葡萄糖
    0.4mM; 4:1
    寡霉素
    DMSO
    50μM/SH测定培养基
    5μM

    抗霉素A
    DMSO
    80μM/SH测定培养基
    8μM

    FCCP
    DMSO
    50μM/SH +丙酮酸钠 5μM

    丙酮酸钠
    -
    100 mM/SH测定培养基
    10 mM
    pH = 7.4,过滤器
     

第二部分。渗透性棕色脂肪细胞呼吸

材料和试剂

  1. D-甘露糖醇(Sigma-Aldrich,目录号:M9647)
  2. 蔗糖(Thermo Fisher Scientific,目录号:S5)
  3. 磷酸二氢钾(KH 2 PO 4)(Thermo Fisher Scientific,目录号:P285)
  4. 六水合氯化镁(MgCl 2)(Thermo Fisher Scientific,目录号:M33)
  5. HEPES(Sigma-Aldrich,目录号:H4034)
  6. 乙二醇 - 双(β-氨基乙基醚)-N,N,N,N'-四乙酸四钠盐(EGTA)(Sigma-Aldrich,目录号:E8145)
  7. 脂肪酸游离BSA(EMD Millipore Corporation,CalBiochem,目录号:126575)
  8. L - ( - ) - 苹果酸(Sigma-Aldrich,目录号:02288)
  9. 琥珀酸酯(Sigma-Aldrich,目录号:S3674)
  10. 寡霉素A(Sigma-Aldrich,目录号:75371)
  11. 来自链霉菌属(Sigma-Aldrich,目录号:A8674)
  12. 羰基氰4-(三氟甲氧基)苯腙(FCCP)(Sigma-Aldrich,目录号:C2920)
  13. 罗望酮(Sigma-Aldrich,目录号:R8875)
  14. (+) - 乙莫克舍钠盐水合物(Sigma-Aldrich,目录号:E1905)
  15. 腺苷5'-二磷酸单钾盐二水合物(Sigma-Aldrich,目录号:A5285)
  16. L - ( - ) - 肉碱(Thermo Fisher Scientific,Acros Organics,目录号:241040100)
  17. 琥珀酸酯(Sigma-Aldrich,目录号:S3674)
  18. L - ( - ) - 苹果酸(Sigma-Aldrich,目录号:02288)
  19. 丙酮酸钠(Sigma-Aldrich,目录号:P5280)
  20. 棕榈酰基-L-肉碱氯化物(Sigma-Aldrich,目录号:P1645)
  21. 棕榈酰辅酶A锂盐(Sigma-Aldrich,目录号:P9716)
  22. 3x MAS缓冲区(参见配方)
  23. XF PMP试剂(Seahorse Bioscience,目录号:102504-100)(参见Recipes)

设备

  1. 与完整的棕色脂肪细胞呼吸协议相同的设备

程序

  1. 准备细胞,如完整的棕色脂肪细胞呼吸协议的步骤1-2所述
  2. 在实验当天,解冻1x MAS和食谱部分1中描述的预制底物溶液。
  3. 在1x MAS中制备10x抑制剂溶液。如果使用脂肪酸底物,请按照配方第2b节中所述新鲜制备
    1. 在装载之前,在CO 2 2-free培养箱中将小柱至少浸泡3小时。
    2. 负载端口:端口A为50μl,端口B为55μl,端口C为60μl,端口D为65μl,校准盒。
    3. 在1x MAS中制备7.5nM PMP,体积足以每孔450μl
    4. 轻轻地用1x MAS缓冲液洗一次细胞。
    5. 完全排空洗涤,加入含有7.5 nM PMP的450μl1x MAS。不需要在CO 2无培养箱中孵育。
    6.  立即加载平板,并用3分钟混合物和2运行海马测定 ?min测量(无等待)。每个条件进行2-3次测量。不要 在抗霉素A注射前超过1小时的测定时间 线粒体完整性将受到影响。

代表数据


图4.用A)琥珀酸盐+ ADP +鱼藤酮,B)寡霉素和C)抗霉素注射的透化的BAT的代表性OCR图

笔记

  1. 确保透化的最简单的方法是检测由膜不可渗透的底物琥珀酸酯引起的呼吸。琥珀酸也可用于滴定最佳XF PMP试剂浓度
  2. 始终加载所有四个进样口。如果不使用一个端口,请加载1x MAS缓冲区。
  3. 每个条件至少使用三个技术重复。
  4. 为了确保数据的质量,确保pH在整个运行期间是稳定的,并且每次测量后氧气张力恢复到基本水平。
  5. 如果OCR超过2,000 pmol/min,请转到海马Excel数据查看器中的"特殊操作"选项卡,单击"更改O2计算方法"并选择"增益(固定)"算法,因为AKOS算法不会处理高OCR数据正确。始终将增益(固定)数据表示为基线测量的百分比。
  6. 有关透化的更多信息,请参阅Divakaruni等人(2014年)和Salabei等人(2014年)。

食谱

  1. 3x MAS缓冲区
    试剂
    集中
    金额1 L MAS
    甘露醇
    660 mM
    120.23克
    蔗糖
    210 mM
    71.88克
    KH 2 PO 4
    30 mM
    4.08克
    MgCl 2
    15 mM
    15ml的1.0M溶液
    HEPES
    6 mM
    加入6ml 1.0M溶液
    EGTA
    3 mM
    12ml 0.25M溶液
    脂肪酸游离BSA
    0.6%(w/v)
    6.0克
    将试剂溶于双蒸水中,用KOH将pH值调至7.2,无菌过滤,并在-20℃下保存 可选:在双蒸水中稀释至1X,用KOH稀释至pH 7.2,无菌过滤,并储存在-20°C
  2. XF PMP试剂
    储液:10μM
    储存:-20°C
    库存解决方案
    试剂
    MW
    集中
    溶剂
    评论
    马拉泰
    134.09  
    1.0 M
    ddH 2 O   
    用KOH将pH调节至7.2。溶液是强酸性的,根据最终体积计算摩尔浓度
    琥珀酸盐
    270.1
    1.0 M
    ddH sub 2 O
    用KOH缓冲至pH7.2
    寡霉素
    791.06
    20 mM
    DMSO
    复合V抑制剂
    抗菌素A
    548.63
    40 mM
    DMSO
    复合物III抑制剂
    FCCP
    254.17
    40 mM
    DMSO
    化学脱离剂
    Rotenone
    394.4
    40 mM
    DMSO
    复合物I抑制剂
    Etomoxel
    338.76
    40 mM
    DMSO
    CPT-1抑制剂阻断棕榈酰辅酶A转化为棕榈酰肉碱
    ADP,K +盐
    501.32
    用作粉末


    L - ( - ) - Carnitine   
    161.2
    用作粉末



    基板
    基板
    MW
    10x浓度。港口注入
    添加剂(10x浓度,用于进样口)
    琥珀酸盐
    270.1
    100 mM
    40mM ADP,50μMrotenone
    马拉泰
    134.09
    30 mM
    40 mM ADP
    丙酮酸
    110.94
    100 mM
    40mM ADP,30mM苹果酸盐
    棕榈酰肉碱
    436.07
    1 mM
    40mM ADP,30mM苹果酸盐,5mM肉碱
    棕榈酰-CoA   1,004.94
    1 mM
    40mM ADP,30mM苹果酸盐,5mM肉碱

    1. 在1×MAS缓冲液中制备琥珀酸盐,苹果酸盐和丙酮酸盐,用KOH调节pH ?至7.2。无菌过滤器,等分试样和存储 在-20°C
      1. 例如,对于5ml琥珀酸盐溶液,加入 0.5ml 1.0M琥珀酸盐,100.2mg ADP,1.67ml 3x MAS和2.83ml ddH 2 O 并用KOH调节pH至7.2。 (在实验当天加入鱼藤酮)
      2. 对于每100μl额外的体积,作为pH滴定的结果,添加50微升3x MAS以保持渗透强度。
      3. 例如,如果5ml琥珀酸盐溶液的最终体积为5.1ml, ?加入50μl3x MAS。这将导致试剂的轻微稀释 其不应显着影响测定。但是,考虑到这一点 可变性,我们建议准备大批量的解决方案和使用 给定项目中所有实验的相同库存。
    2. 对于脂肪 ?酸,制备添加剂(除肉碱外)在1x MAS中的溶液 缓冲液中,并且在实验日之前用KOH调pH至7.2。无菌 过滤,等分,并储存在-20°C。添加肉碱和脂肪酸 溶液在实验当天。

致谢

渗透性棕色脂肪细胞呼吸方案改编自Divakaruni等人(2014)。这项工作由国家卫生研究院授予R01DK074778,R01DK56690-11和由瑞典研究委员会以及DIABAT FP7,协作EU格兰特HEALTH-F2-2011-278373和Wallenberg基金会支持。

参考文献

  1. Cannon,B。和Nedergaard,J。(2001)。 来自棕色脂肪组织和克隆棕色脂肪细胞样细胞系的脂肪前体细胞培养物。/a> Methods Mol Biol 155:213-224
  2. Divakaruni,A.S.,Rogers,G.W.and Murphy,A.N。(2014)。 使用海马XF分析仪或克拉克型氧电极测量透化细胞中的线粒体功能。 a> Curr Protoc Toxicol 60:25 22 21-25 22 16.
  3. Salabei,J.K.,Gibb,A.A。和Hill,B.G。(2014)。 使用细胞外通量分析来综合测量透化细胞中的呼吸活性。 Nat Protoc 9(2):421-438。
  4. 这些研究结果表明,这些研究结果表明,该方法可以有效地解决这些问题, 。 激素诱导的线粒体分裂被棕色脂肪细胞用作能量消耗的放大途径。/a> EMBO J 33(5):418-436
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