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Mar 2018

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Using Stable Isotopes in Bone Marrow Derived Macrophage to Analyze Metabolism
利用稳定同位素研究骨髓源性巨噬细胞的代谢   

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Abstract

Using gas chromatography mass spectrometry (GC-MS) to analyze the citric acid cycle (CAC) and related intermediates (such as glutamate, glutamine, GABA, and aspartate) is an analytical approach to identify unexpected correlations between apparently related and unrelated pathways of energy metabolism. Intermediates can be as expressed as their absolute concentrations or relative ratios by using known amounts of added reference standards to the sample. GC-MS can also distinguish between heavy labeled molecules (2H- or 13C-labeled) and the naturally occurring most abundant molecules. Applications using tracers can also assess the turnover of specific metabolic pools under various physiological and pathological conditions as well as for pathway discovery.

The following protocol is a relatively simple method that is not only sensitive for small concentrations of metabolic intermediates but can also be used in vivo or in vitro to determine the integrity of various metabolic pathways, such as flux changes within specific metabolite pools. We used this protocol to determine the role of phosphoenolpyruvate carboxykinase 1 (Pck1) gene in mouse macrophage cells to determine the percent contribution from a precursor of 13C labeled glucose into specific CAC metabolite pools.

Keywords: Macrophage (巨噬细胞), Bone marrow-derived macrophages (骨髓源性巨噬细胞), Immunology (免疫学), Stable isotopes (稳定同位素), Metabolism (代谢), Macrophage polarization (巨噬细胞极化), Stable isotopes (稳定同位素), Chromatography mass spectrometry methods (色谱质谱连用分析法), GC-MS (气相色谱质谱连用分析法)

Background

With the development of altered gene expression in cells and mice, there is a need to understand how these deleted or over-expressed genes impact the regulation of metabolic pathways. In this protocol, we used stable isotopes to determine how the flux of glucose into the CAC altered the contribution of glucose into the pools of citrate, succinate and malate. The use of stable isotopes with targeted analysis of metabolism is just one benefit to using stable isotopes in cell culture.

The method described in this protocol for functional quantification of intracellular metabolites was done by growing bone marrow-derived macrophage cells (BMDM) in U-13C-glucose medium. The cells were extracted in organic solvent and the percent contribution of 13C glucose was calculated. The fractional amount of 13C label incorporated into each the CAC-related metabolite pools was also determined. The calculations were based on the ratio of 13C label of each intracellular metabolite versus the unlabeled metabolite; for absolute concentration analysis of cell samples, one would need to correct for reference to the intracellular volume of the extracted cells (Feldberg et al., 2009), as well as add non-interfering reference standards for quantifying of levels of CAC intermediates, as previously described (Ko et al., 2018). This protocol can also be used in isolated perfused livers or whole body metabolism studies (Yang et al., 2008a; Zhang et al., 2015).

Using a novel mouse model that had a deletion of phosphoenolpyruvate carboxykinase 1 (Pck1) in the myeloid cells (Pck1MC-KO), stable isotopes were used to determine the role of this gene in macrophages (Ko et al., 2018) with respect to glucose metabolism. The protocol explains the isolation and differentiation of BMDM. These cells were isolated, differentiated and incubated with U13C-glucose to analyze their metabolism. The BMDM cells were collected, and the fractional contribution of the precursor to the product was based on the mole percent enrichments (MPE) derived from the 13C label incorporation into the total pool of each of the metabolites (products). Mass isotopomer analysis enables the measurements of unlabeled analyte (M0) relative to the labeled analyte (M+1, 2, or 3, etc.), (Yang et al., 2008a; Kombu et al., 2011; Ko et al., 2018). The measured mass isotopomer distributions were calculated for each of the masses and expressed as mole percent enrichment (MPE) after correcting for natural isotope abundances.

Materials and Reagents

  1. FisherbrandTM sterile 100 mm x 15 mm polystyrene Petri dish (Fisher Scientific, Fisher ScientificTM, catalog number: FB0875713 )
  2. Costar® TC-Treated 6-well Plates (Corning, catalog number: CLS3506 )
  3. 15 ml conical tubes (SARSTEDT, catalog number: 62.554.502 )
  4. Sterile individually packaged 5 ml pipettes (SARSTEDT, catalog number: 86.1253.001 )
  5. Sterile 1 ml syringe with 26 G needle (Fisher Scientific, catalog number: 14-829-6A)
    Manufacturer: BD, catalog number: 305537 .
  6. 10 ml syringes (Thermo Fisher Scientific, catalog number: S7515-10 )
  7. BD Precisionglide® syringe needles, gauge 23, L3/4 in. (BD, catalog number: 305143 )
  8. BD Precisionglide® syringe needles, gauge 18, L 1 in. (BD, catalog number: 305195 )
  9. Cell strainer, 70 μm, sterile (Corning, catalog number: 352350 )
  10. 50 ml conical tube (SARSTEDT, catalog number: 62.547.254 )
  11. Disposable Borosilicate Glass tubes 16 mm x 125 mm (Globe Scientific, catalog number: 1515 )
  12. LysM-specific Pck1 knock-out mice (Pck1MC-KO mice)
    Note: The mice were generated by crossing Pck1flox/flox mice with LysM-Cre+/− transgenic mice expressing Cre-recombinase under control of the LysM promoter. Pck1flox/flox mice were used as controls. All mice are in the C57Bl/6J background. All animals were housed in a temperature-controlled facility with a 12 h light/dark cycle in compliance with the Institutional Animal Care and Use Committee (IACUC) of Case Western Reserve University.
  13. Lipopolysaccharides from Escherichia coli 026:B6 (Sigma-Aldrich, catalog number: L8274 )
  14. IL-4, Animal-component free, recombinant, expressed in E. coli (Sigma-Aldrich, catalog number: SRP3211 )
  15. Ethanol Solution 70%, Molecular Biology Grade (Fisher Scientific, Fisher BioagentsTM, catalog number: BP8201500
  16. FBS (fetal bovine serum) (Fisher Scientific, FisherbrandTM, catalog number: 03-600-511 )
  17. Dulbecco's modified Eagle Medium (DMEM) high glucose, pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11995065 )
  18. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 10570063 )
  19. L-glutamine GlutaMAX (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  20. Recombinant Murine Macrophage Colony-stimulating factor (PeproTech, catalog number: 315-02 )
  21. Glucose (Sigma-Aldrich, catalog number: G8270 )
  22. Lipopolysaccharide (LPS) from E. coli 02:B6 (Sigma-Aldrich, catalog number: L8274 )
  23. Interleukin-4 (IL-4) (PeproTech, catalog number: 214-14 )
  24. [13C]-succinate [Sodium bis (2-ethylhexyl) sulfo (succinate-13C4)] (98%) (Sigma-Aldrich, catalog number: 719269 )
  25. [13C]-malate (Sigma-Aldrich, catalog number: 750484 )
  26. [13C]-citrate (Sigma-Aldrich, catalog number: 492078 )
  27. Methanol HPLC (Sigma-Aldrich, catalog number: 1005706 )
  28. BSTFA + 10% TMCS-Regisil® (Regis Technologies, CAS: 25561-30-2; 75-77-4)
  29. N-Methyl-N-(t-butyldimethylsilyl) trifluoroacetamide (TBDMS) (Sigma-Aldrich, catalog number: 394882-25ML )
    Note: If using TMCS as a derivative, see references Yang et al. (2008a); Kombu et al. (2011); Ko et al. (2018). 
  30. Acetic acid, Glacial (Certified ACS) Fisher Chemical (Fisher Scientific, catalog number: A38-212)
    Manufacturer: Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FLA38212 .
  31. Methanol, OptimaTM LC/MS Grade, Fisher Chemical (Fisher Scientific, Thermo ScientificTM, catalog number: A456-1 )
  32. Water, OptimaTM LC/MS Grade, Fisher Chemical (Fisher Scientific, Thermo ScientificTM, catalog number: W64 )
  33. QuickStart TM Bradford Protein Assay Kit 1 (Bio-Rad Laboratories, catalog number: 5000201 )
  34. Generate macrophage differentiation media (MDM) (see Recipes)
  35. 5% acetic acid in methanol/water (1:1) extraction buffer (see Recipes) 
  36. LPS stock solution (see Recipes)
  37. IL-4 stock solution (see Recipes)

Equipment

  1. Kelly Forceps, Box Lock, Straight Steel (Grainger, catalog number: 4WPD9 )
  2. Sterile cell scraper (Fisher Scientific, FisherbrandTM, catalog number: 08-100-241 )
  3. GC vial cap, 9 mm (Agilent Technologies, catalog number: 5182-0717 )
  4. GC vial, 2 ml (Agilent Technologies, catalog number: 5181-3375 )
  5. Thermo ScientificTM NalgeneTM Polypropylene Graduated Cylinders (Fisher Scientific, catalog number: 08-572D)
    Manufacturer: Thermo Fisher Scientific, Thermo ScientificTM, catalog number: N36620100 .
  6. Dumont forceps #5 (Fine Science Tools, catalog number: 11252-20 )
  7. Dumont forceps #55 (Fine Science Tools, catalog number: 11255-20 )
  8. Dumont forceps AA (Fine Science Tools, catalog number: 11210-20 )
  9. Tissue culture hood (Thermo Fisher Scientific, catalog number: 51022482 )
  10. Refrigerated tabletop centrifuge for 15-50 ml conical tubes (Eppendorf, model: 5430R )
  11. 37 °C, 5% CO2 water-jacketed incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: 3110 )
  12. P20 pipetman (Gilson, catalog number: F123600 )
  13. P200 pipetman (Gilson, catalog number: F123601 )
  14. P1000 pipetman (Gilson, catalog number: F123602 )
  15. Homogenizer for tissues (IKA, model: T25 digital ULTRA-TURRAX®, catalog number: 0003725001 )
  16. Dry Block Heater (VWR, catalog number: 75838-282 )
  17. Frigidaire 13.8 cu. Ft. Frost Free Upright Freezer (The Home Depot) 
  18. Large Series, Single Door, Hinged Autoclave (Consolidated Sterilizer Systems, model: LR-36E )
  19. Basic Laboratory Hoods (Labconco, model: 2246500
  20. Inverted light microscope (Leica Microsystems, model: DM IL LED )
  21. GC-MS (Agilent Technologies, model: 5973-MSD ) equipped with an Agilent 6890 GC system 
  22. DB-17MS capillary column, 30 m x 0.25 mm x 0.25 μm (Agilent Technologies)

Procedure

  1. Isolation and differentiation of Bone Marrow-Derived Macrophages and incubation with stable isotope
    1. Harvesting Marrow from Mouse Femur and Tibia
      1. Prepare macrophage differentiation media (MDM) (see Recipes). Warm up the media in a 37 °C water bath for 30 min before harvesting the marrow. 
      2. Euthanize the mouse with CO2 asphyxiation and cervical dislocation and place the mouse in a beaker with 100% ethanol. 
      3. Using sterile scissors in a tissue culture hood, clip the skin and remove the skin from the lower part of the mouse body.
      4. Remove the legs from the mouse body with scissors that have been placed in a beaker with 100% ethanol (Figure 1). 
      5. Remove all remaining hair and skin. Remove muscle, and fat surrounding the bones. Place the bones in 100% ethanol for 5 min. Pour 8 ml of MDM into a sterile 100 mm x 15 mm polystyrene Petri dish. Transfer the bones from the 100% ethanol to the Petri dish with media. 
      6. Separate at the knee joint the femoral bones from the tibia and fibula. Be careful not to break the bones. 
      7. Cut off each end of the bone as shown in Figure 1. You should have a total of 3 bones for each mouse leg in one Petri dish with media.


        Figure 1. Isolating bone marrow from mouse tibia, fibula and femur. The mouse was euthanized. A. Image illustrating where to cut and remove the leg. B. Schematic diagram of the positions to be cut at mouse leg for isolating bone marrow. Cut the legs at position 1 (red dashed lines). Next remove the fur, skin and muscle from the leg. Then cut the bones at the red dotted lines to isolate the bones in the order indicated (numbers 2, 3, 4 and 5). Note that each bone is cut medially and distally. 

      8. Fill 26 G needle/1 ml syringe with macrophage differentiation media (MDM) (see Recipes). Put the tip of the needle into the bone and flush the bone marrow from both ends of the bone (Figure 2). Do this for the femur, fibula and tibia. Repeat flushing the marrow out of the bones until the bones turn white.


        Figure 2. Flushing the bones. A. Image showing the marrow compartment in the bone. B and C. The femur before (B) flushing and after (C) flushing (see Video 1).

        Video 1. Flushing of the bones to remove bone marrow

      9. Collect the cells in MDM and pass them through a 70 μm cell strainer into a sterile conical tube. This removes debris that may contaminate your cells (see Video 2).

        Video 2. Removing debris from isolated cells

      10. Spin down cells at 1,950 x g for 5 min at 4 °C. Count the cells and resuspend the pellets with macrophage differentiation media (MDM) so that you can seed 5 x 106 cells and MDM into each well of the 6-well plate.
      11. Incubate plates for 7 days at 37 °C and 5% CO2. Change media every three days with freshly made media.
    2. Differentiation of macrophage to M1 or M2
      When BMDM are almost confluent (at Day ~7), replace with new MDM. In order to polarize the macrophages, we used either lipopolysaccharide (LPS) or interleukin-4 (IL-4).
      1. For classic M1 activation: Add lipopolysaccharide (LPS) at a dose of 100 ng/ml (10 μl of stock solution into 4 ml MDM) and incubate for 4 h.
      2. For classic M2 activation: Add interleukin-4 (IL-4) at a dose of 40 ng/ml (1.6 μl of stock solution into 4 ml of MDM) and incubate for 4 h.
    3. Metabolism studies using U-13C Glucose
      1. Replace the MDM in the differentiated macrophages (already treated with either LPS or IL-4) with medium that contains no glucose, 10% charcoal-stripped FBS, 100 ng/ml LPS, 5 mM U-13C glucose (99.98% enriched) and incubate overnight at 37 °C. We did this because the gene of being investigated, Pck1, is activated in the fasted state, so we removed the glucose and insulin. 
      2. At the next day, collect the media and cells. To collect media, pipet off half of the media. Keep 1 ml of the media for measurement of isotope enrichment. To collect cells, scrape the wells to dislodge the cells. Pipet up the cells and remaining media and place into conical tubes. Spin down the cells at 1,950 x g for 5 min at 4 °C and resuspend the pellets in 50 μl of 1x PBS. Save 20 μl for measurement of protein concentration by Bradford assay. Freeze sample at -20 °C to lyse cells. The protein concentrations are used for normalization in the metabolite analysis. Cells are for analysis by GC-MS. In our case, we sent the cells to the Case Western Reserve University Mouse Metabolic Phenotyping Center (MMPC) for analysis by GC-MS.
      3. Bradford assay: Carry out the assay according to the manufacturer’s directions (Quick StartTM Bradford Protein Assay). We used 2 mg/ml BSA as a standard. Pipette 20 μl of the resuspended cells into a 1.5 ml Eppendorf tube and add 1 ml of 1x Dye reagent from the Quick StartTM Bradford Protein Assay. Mix the sample and incubate at room temperature for 5 min. Place the sample in a cuvette. Set the spectrophotometer to 595 nm. Zero the instrument with the blank sample (100 μl water and 1 ml of 1x Dye reagent). Measure the absorbance of the BSA standards and the unknown (macrophage cells). Determine the amount of protein in the sample from the standard curve (if 2 μl aliquot of sample yields a 595 nm value equivalent to 250 μg/ml of protein, then the cell sample would have a protein concentration of 2.5 mg/ml).
      4. For the study on Pck1MC-KO macrophages, the cells were sent to the Case Western Reserve University Mouse Metabolic Phenotyping Center (MMPC) for analysis by GC-MS. The following section displays an example for the study of metabolites in the LPS-activated macrophage. The purpose of the example study was to compare the metabolites in Pck1MC-KO (experimental) and Pck1fl/fl (control) macrophages after a 24 h treatment of LPS.

  2. Analysis of the citric acid cycle (CAC) and related intermediates (such as citrate, succinate and malate) using gas chromatography mass spectrometry (GC-MS)
    1. Cell sample preparation
      Briefly, this approach utilizes the rapid reaction of silylating reagents with alcohols, acids and amines to form silyl derivatives (Yang et al., 2008a and 2008b; Kombu et al., 2011; Zhang et al., 2015). Commercial silylating reagents are available as combinations to accelerate the reaction, as well as to react with the hindered group. For example either a mixture of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and trimethylchlorosilane (TMCS) to form a TMS derivative, or a mixture of N-Methyl-N-(t-butyldimethylsilyl)-trifluoroacetamide (MTBSTFA) and t-butyldimethylchlorosilane (TBDMS) to form TBDMS derivatives can be used (Yang et al., 2008a and 2008b; Kombu et al., 2011; Zhang et al., 2015).
      1. For absolute quantification of analytes, as previously described (Ko et al., 2018): 50 μl of cells are spiked with selected internal reference standards such as ~5 nmole of each metabolite [13C6] citrate, [13C4] succinate, and/or [2H4]-3-hydroxyglutarate.
        Note: Choice of internal reference standards are dependent on precursor tracer used such that there is no interference of the M+ labeling from the tracer versus the labeled standards added; its best to use non-endogenously produced reference standards, such as tricarballylic acid. 
      2. Homogenize the cells with 3-5 ml of 5% acetic acid in methanol/water (1:1) extraction buffer (chilled on ice) for 2 min on ice bath.
      3. Centrifuge the homogenate at 670 x g for 30 min at 4 °C. Decant the supernatant into a glass test tube and save on ice and process immediately
        Note: May freeze the supernatant at -80 °C until assaying for GC-MS.
      4. TMSC/TBDMS derivatization procedure: pipette 100-200 μl of the supernatant collected in Steps B1b and B1c into 16 mm x 125 mm disposable borosilicate glass tubes.
      5. Dry completely under nitrogen gas (Figure 3).


        Figure 3. Nitrogen Dryer

      6. React by adding 70 μl of either derivatizing TBDMS reagents (in a pyridine-based solvent) and heat on a heating block at 80 °C for 1 h (Figure 4).
        Note: May react at room temperature overnight.


        Figure 4. Heat block with tubes for derivatization

      7. Transfer to GC insert and cap (Figure 5); follow GC parameters and the monitored SIM ions (m/z) for each analyte, as outlined (Yang et al., 2008a and 2008b; Kombu et al., 2011; Zhang et al., 2015).


        Figure 5. Samples put into GC tubes

    2. GC-MS Analysis
      1. TMSC or TBDMS derivatives are analyzed using an Agilent 5973-MSD equipped with an Agilent 6890 GC system, and a DB-17MS capillary column (30 m x 0.25 mm x 0.25 μm; may use 60 m). The mass spectrometer is operated under electron impact mode (EI) or ammonia chemical ionization mode (CI) and selective ion monitoring (SIM) m/z for each analyte; When stable isotopes are applied, the SIM for M0-M+ m/z are monitored (Yang et al., 2008a and 2008b; Kombu et al., 2011; Zhang et al., 2015) (Figure 6).


        Figure 6. GC-MS apparatus used in this protocol

      2. After the GC-MS run, calculate the area under the curve for succinate, citrate and malate and the ratios to the internal standard (Table 1). After background correction, calculate the MPE% using the following formula:
        MPE% = [(13C labeled M1 thru M + for each of the CAC intermediates) x (13C labeled + unlabeled) - 1] x 100 (Table 1)

        Table 1. The calculation of mole percent enrichments from 13C-glucose into citrate, succinate and malate. M0, M1 to M3 were calculated based on the ratio of labeled intracellular metabolites to unlabeled and were corrected with reference to the intracellular volume of the extracted cells (Feldberg et al., 2009). The %MPE was calculated as %m2 = [(13C labeled M1 thru M + for each of the CAC intermediates) x (13C labeled + unlabeled) - 1] x 100.

Data analysis

  1. For experimental design, the stable isotope studies are done in triplicate for each sample. The triplicates are considered an n = 1. All experiments are done for a total of n = 6 times.
  2. MPE of M1 to M3 was calculated based on the ratio of labeled intracellular metabolites to unlabeled and corrected with reference to the intracellular volume of the extracted cells (Feldberg et al., 2009). The %MPE was calculated as MPE% = [(13C labeled M1 thru M+ for each of the CAC intermediates) x (13C labeled + unlabeled)] x 100.
  3. For statistical analysis, we used GraphPad Prism. To compare two data sets (Pck1flox/flox and Pck1MC-KO mice), we performed a Student's t-test (Figure 7).


    Figure 7. Contribution of glucose to the central metabolism of LPS-activated macrophages. BMDMs isolated from Pck1flox/flox and Pck1MC-KO mice were incubated with [U-13C] glucose in the presence of LPS for 16 h, and GC-MS was performed for the calculation of the contribution of 13C to M2-CAC. Values represent the means ± SEM for n = 6 per group. *P < 0.05 compared to Pck1flox/flox group.

Notes

  1. Internal standards are necessary due to variation in each run. For this experiment, the samples (n = 6 per group) were all run on the GC-MS at the same time to reduce variation.
  2. Isolation of BMDM was done for each animal one at a time. For example, one Pck1flox/flox mouse was euthanized, legs dissected and bone marrow was flushed from the femur tibia and fibula. The cells were kept cold on ice. Then the next animal was euthanized and so on. The cells were kept on ice until all of the bone marrow was collected.
  3. Media was prewarmed before plating of BMDM.
  4. To study the initiation of signaling pathways (e.g., TLR4 signaling in M1 activation or IL-4R signaling in M2 activation) that require rapid turnover, cells are suggested to be treated with higher dose of stimulus (e.g., 100 ng/ml LPS or 20 ng/ml IL-4) within 30 min; to determine the metabolic pathways (e.g., citric acid cycle and related intermediates) using isotopomers, 16-24 h incubation with lower dose of stimulus (e.g., 20 ng/ml LPS or 10 ng/ml IL-4) is suggested.
  5. Macrophages undergo morphological changes during their activation. Compared to the non-stimulated macrophage with rounded shapes, LPS-treated macrophages display pancake-like shape within 24 h of stimulation. On the other hand, IL-4-treated cells promote cell elongation.
  6. The cells isolated from the bone marrow will adhere to the tissue culture plates within 24 h-48 h. The MDM contains M-CSF that will allow for myeloid cells to be differentiated into macrophages. If the cells continue to float in the media there may be contamination of the cells. If this occurs, you will need fresh media and re-isolate the bone marrow cells.

Recipes

  1. Generate macrophage differentiation media (MDM)
    Combine 500 ml of sterile Dulbecco’s modified Eagle Medium (DMEM) with:
    55 ml of 10% fetal bovine serum (FBS) to the DMEM
    5 ml of 1x glutamine to DMEM
    5 ml of 1x Penicillin-Streptomycin (10,000 U/ml Pen, 10 mg/ml Strep)
    5 μg of macrophage colony-stimulating factor (M-CSF) to 500 ml of DMEM
  2. 5% acetic acid in methanol/water (1:1) extraction buffer (chilled on ice)
    Make methanol/water by adding 50 ml of methanol to 50 ml of dH2O to make 100 ml
    In a separate graduated cylinder, combine 5 ml of acetic acid with 95 ml of the methanol/water made above
  3. LPS stock solutions
    Make up to 40 µg/µl in sterile water and aliquot
    Store at -80 °C
  4. IL-4 stock solution
    Make up to 100 µg/µl in sterile water and aliquot
    Store at -80 °C

Acknowledgments

The Case MMPC was supported by NIH grant U24 DK76174. The complete study on Pck1MC-KO mice has been published (Ko et al., 2018).

Competing interests

The authors declare that they have no conflicts of interest with the contents of this article.

Ethics

All animal experiments were approved by Case Western Reserve University Institutional Animal Care and Use Committee (IACUC).

References

  1. Feldberg, L., Venger, I., Malitsky, S., Rogachev, I. and Aharoni, A. (2009). Dual labeling of metabolites for metabolome analysis (DLEMMA): A new approach for the identification and relative quantification of metabolites by means of dual isotope labeling and liquid chromatography-mass spectrometry. Anal Chem 81(22): 9257-9266.
  2. Ko, C. W., Counihan, D., Wu, J., Hatzoglou, M., Puchowicz, M. A. and Croniger, C. M. (2018). Macrophages with a deletion of the phosphoenolpyruvate carboxykinase 1 (Pck1) gene have a more proinflammatory phenotype. J Biol Chem 293(9): 3399-3409.
  3. Kombu, R. S., Brunengraber. H. and Puchowicz, M. A. (2011). Analysis of the citric acid cycle intermediates using gas chromatography-mass spectrometry. In: Metz, T. O. (Ed.) Metabolic Profiling: Methods and Protocols. Humana Press 147-157.
  4. Yang, L., Kasumov, T., Kombu, R. S., Zhu, S. H., Cendrowski, A. V., David, F., Anderson, V. E., Kelleher, J. K. and Brunengraber, H. (2008a). Metabolomic and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle: II. Heterogeneity of metabolite labeling pattern. J Biol Chem 283(32): 21988-21996.
  5. Yang, L., Kombu, R. S., Kasumov, T., Zhu, S. H., Cendrowski, A. V., David, F., Anderson, V. E., Kelleher, J. K. and Brunengraber, H. (2008b). Metabolomic and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle. I. Interrelation between gluconeogenesis and cataplerosis; formation of methoxamates from aminooxyacetate and ketoacids. J Biol Chem 283(32): 21978-21987.
  6. Zhang, Y., Zhang, S., Marin-Valencia, I. and Puchowicz, M. A. (2015). Decreased carbon shunting from glucose toward oxidative metabolism in diet-induced ketotic rat brain. J Neurochem 132(3): 301-312.

简介

使用气相色谱质谱(GC-MS)分析柠檬酸循环(CAC)和相关中间体(如谷氨酸,谷氨酰胺,GABA和天冬氨酸)是一种分析方法,用于识别明显相关和不相关的能量途径之间的意外相关性代谢。通过使用已知量的样品添加的参考标准,中间体可以表示为它们的绝对浓度或相对比例。 GC-MS还可以区分重标记分子( 2 H-或 13 C-标记的)和天然存在的最丰富的分子。使用示踪剂的应用还可以评估在各种生理和病理条件下以及用于途径发现的特定代谢池的周转。

以下方案是一种相对简单的方法,不仅对小浓度的代谢中间体敏感,而且还可以 in vivo 或 in vitro 用于确定各种新陈代谢的完整性途径,如特定代谢物池内的通量变化。我们使用该协议来确定磷酸烯醇丙酮酸羧激酶1 ( Pck1 )基因在小鼠巨噬细胞中的作用,以确定 13 C将葡萄糖标记为特定的CAC代谢物库

【背景】随着细胞和小鼠中基因表达改变的发展,需要了解这些缺失或过表达的基因如何影响代谢途径的调节。在该方案中,我们使用稳定同位素来确定进入CAC的葡萄糖通量如何改变葡萄糖对柠檬酸盐,琥珀酸盐和苹果酸盐的贡献。使用稳定同位素和目标分析代谢只是在细胞培养中使用稳定同位素的一个好处。

本方案中描述的用于细胞内代谢物功能定量的方法是通过在U- 13 C-葡萄糖培养基中培养骨髓衍生的巨噬细胞(BMDM)来完成的。在有机溶剂中提取细胞,计算 13 C葡萄糖的百分比贡献。还测定了掺入每个CAC相关代谢物库中的 13 C标记的分数量。计算基于每种细胞内代谢物与未标记代谢物的 13 C标记的比率;对于细胞样品的绝对浓度分析,需要校正提取细胞的细胞内体积(Feldberg et al。,2009),并添加非干扰参考标准用于定量CAC中间体的水平,如前所述(Ko et al。,2018)。该方案也可用于分离的灌注肝脏或全身代谢研究(Yang et al。,2008a; Zhang et al。,2015)。

使用在骨髓细胞中删除磷酸烯醇丙酮酸羧激酶1(Pck1)的新型小鼠模型(Pck1 MC-KO ),使用稳定同位素来确定其作用。关于葡萄糖代谢的巨噬细胞中的这种基因(Ko et al。,2018)。该协议解释了BMDM的分离和分化。分离这些细胞,分化并与U 13 C-葡萄糖一起温育以分析它们的代谢。收集BMDM细胞,并且前体对产物的分数贡献基于来自 13 C标记物掺入每种代谢物的总库中的摩尔百分比富集(MPE)(产品)。质量同位素异构体分析能够测量未标记分析物(M0)相对于标记分析物(M + 1,2或3,等),(Yang et al。 ,2008a; Kombu et al。,2011; Ko et al。,2018)。计算每个质量的测量的质量同位素异构体分布,并在校正天然同位素丰度后表示为富集的摩尔百分比(MPE)。

关键字:巨噬细胞, 骨髓源性巨噬细胞, 免疫学, 稳定同位素, 代谢, 巨噬细胞极化, 稳定同位素, 色谱质谱连用分析法, 气相色谱质谱连用分析法

材料和试剂

  1. Fisherbrand TM 无菌100 mm x 15 mm聚苯乙烯培养皿(Fisher Scientific,Fisher Scientific TM ,目录号:FB0875713)
  2. Costar ® TC处理的6孔板(Corning,目录号:CLS3506)
  3. 15毫升锥形管(SARSTEDT,目录号:62.554.502)
  4. 无菌独立包装5毫升移液器(SARSTEDT,目录号:86.1253.001)
  5. 带有26 G针头的无菌1 ml注射器(Fisher Scientific,目录号:14-829-6A)
    制造商:BD,目录号:305537。
  6. 10 ml注射器(Thermo Fisher Scientific,目录号:S7515-10)
  7. BD Precisionglide ®注射器针头,23号,L3 / 4英寸(BD,目录号:305143)
  8. BD Precisionglide ®注射器针头,18号,L 1英寸(BD,目录号:305195)
  9. 细胞过滤器,70μm,无菌(Corning,目录号:352350)
  10. 50毫升锥形管(SARSTEDT,目录号:62.547.254)
  11. 一次性硼硅酸盐玻璃管16 mm x 125 mm(Globe Scientific,目录号:1515)
  12. LysM特异性Pck1敲除小鼠(Pck1 MC-KO 小鼠)
    注意:小鼠是通过将Pck1 flox / flox 小鼠与表达Cre重组酶的LysM-Cre +/-转基因小鼠杂交而产生的。控制LysM启动子。 Pck1 flox / flox 小鼠用作对照。所有小鼠都处于C57Bl / 6J背景中。根据凯斯西储大学的机构动物护理和使用委员会(IACUC),将所有动物饲养在温度控制的设施中,光照/黑暗周期为12小时。
  13. 来自 Escherichia coli 026:B6的脂多糖(Sigma-Aldrich,目录号:L8274)
  14. IL-4,不含动物成分,重组,以 E表示。大肠杆菌(Sigma-Aldrich,目录号:SRP3211)
  15. 乙醇溶液70%,分子生物学等级(Fisher Scientific,Fisher Bioagents TM ,目录号:BP8201500)&nbsp;
  16. FBS(胎牛血清)(Fisher Scientific,Fisherbrand TM ,目录号:03-600-511)
  17. Dulbecco的改良Eagle培养基(DMEM)高葡萄糖,丙酮酸(Thermo Fisher Scientific,Gibco TM ,目录号:11995065)
  18. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM ,目录号:10570063)
  19. L-谷氨酰胺GlutaMAX(Thermo Fisher Scientific,Gibco TM ,目录号:25030081)
  20. 重组小鼠巨噬细胞集落刺激因子(PeproTech,目录号:315-02)
  21. 葡萄糖(西格玛奥德里奇,目录号:G8270)
  22. 来自 E的脂多糖(LPS)。大肠杆菌 02:B6(Sigma-Aldrich,目录号:L8274)
  23. 白细胞介素-4(IL-4)(PeproTech,目录号:214-14)
  24. [ 13 C] - 琥珀酸[双(2-乙基己基)磺酸钠(琥珀酸 - 13 C 4 ]](98%)(Sigma -Aldrich,目录编号:719269)
  25. [ 13 C] - 苹果酸(Sigma-Aldrich,目录号:750484)
  26. [ 13 C] - 柠檬酸(Sigma-Aldrich,目录号:492078)
  27. 甲醇HPLC(Sigma-Aldrich,目录号:1005706)
  28. BSTFA + 10%TMCS-Regisil ®(Regis Technologies,CAS:25561-30-2; 75-77-4)
  29. N-甲基-N-(叔丁基二甲基甲硅烷基)三氟乙酰胺(TBDMS)(Sigma-Aldrich,目录号:394882-25ML)
    注意:如果使用TMCS作为衍生物,请参阅参考文献Yang et al。,2008a; Kombu等,2011; Ko et al。,2018。&nbsp;
  30. 乙酸,冰川(认证ACS)Fisher化学(Fisher Scientific,目录号:A38-212)
    制造商:Thermo Fisher Scientific,Thermo ScientificTM,目录号:FLA38212。
  31. 甲醇,Optima TM LC / MS级,Fisher Chemical(Fisher Scientific,Thermo Scientific TM ,目录号:A456-1)
  32. 水,Optima TM LC / MS级,Fisher Chemical(Fisher Scientific,Thermo Scientific TM ,目录号:W64)
  33. QuickStart TM Bradford蛋白质分析试剂盒1(Bio-Rad Laboratories,目录号:5000201)
  34. 产生巨噬细胞分化培养基(MDM)(见食谱)
  35. 5%乙酸/甲醇/水(1:1)萃取缓冲液(见食谱)&nbsp;
  36. LPS储备液(见食谱)
  37. IL-4储备液(见食谱)

设备

  1. Kelly镊子,箱锁,直钢(Grainger,目录号:4WPD9)
  2. 无菌细胞刮刀(Fisher Scientific,Fisherbrand TM ,目录号:08-100-241)
  3. GC样品瓶盖,9 mm(Agilent Technologies,目录号:5182-0717)
  4. GC样品瓶,2 ml(Agilent Technologies,目录号:5181-3375)
  5. Thermo Scientific TM Nalgene TM 聚丙烯分度圆柱体(Fisher Scientific,目录号:08-572D)
    制造商:Thermo Fisher Scientific,Thermo Scientific TM ,目录号:N36620100。
  6. Dumont镊子#5(精细科学工具,目录号:11252-20)
  7. Dumont镊子#55(精细科学工具,目录号:11255-20)
  8. Dumont forceps AA(精细科学工具,目录号:11210-20)
  9. 组织培养罩(Thermo Fisher Scientific,目录号:51022482)
  10. 冷藏式台式离心机,适用于15-50 ml锥形管(Eppendorf,型号:5430R)
  11. 37°C,5%CO 2 水夹套培养箱(Thermo Fisher Scientific,Thermo Scientific TM ,型号:3110)
  12. P20移液器(Gilson,产品目录号:F123600)
  13. P200移液器(Gilson,目录号:F123601)
  14. P1000移液器(Gilson,产品目录号:F123602)
  15. 用于组织的均质器(IKA,型号:T25数字ULTRA-TURRAX ®,目录号:0003725001)
  16. 干式加热器(VWR,目录号:75838-282)
  17. Frigidaire 13.8立方米。英尺。 Frost Free立式冰柜(The Home Depot)&nbsp;
  18. 大系列,单门,铰链式高压灭菌器(综合灭菌器系统,型号:LR-36E)
  19. 基本实验室抽油烟机(Labconco,型号:2246500)&nbsp;
  20. 倒置光学显微镜(Leica Microsystems,型号:DM IL LED)
  21. GC-MS(Agilent Technologies,型号:5973-MSD)配备Agilent 6890 GC系统&nbsp;
  22. DB-17MS毛细管柱,30 m x 0.25 mm x0.25μm(安捷伦科技)

程序

  1. 骨髓源性巨噬细胞的分离和分化及稳定同位素孵育
    1. 从小鼠股骨和胫骨收获骨髓
      1. 准备巨噬细胞分化培养基(MDM)(参见食谱)。将培养基在37°C水浴中预热30分钟,然后收获骨髓。&nbsp;
      2. 用CO 2 窒息和颈椎脱位对小鼠实施安乐死,并将小鼠置于含有100%乙醇的烧杯中。&nbsp;
      3. 在组织培养罩中使用无菌剪刀,夹住皮肤并从小鼠身体的下部去除皮肤。
      4. 用放在100%乙醇烧杯中的剪刀从鼠体上取下腿(图1)。&nbsp;
      5. 去除所有剩余的头发和皮肤。去除骨骼周围的肌肉和脂肪。将骨头置于100%乙醇中5分钟。将8毫升MDM倒入无菌的100毫米×15毫米聚苯乙烯培养皿中。用培养基将骨头从100%乙醇转移到培养皿中。&nbsp;
      6. 在膝关节处将股骨与胫骨和腓骨分开。小心不要打破骨头。&nbsp;
      7. 切断骨头的每一端,如图1所示。在一个带有培养基的培养皿中,每个鼠腿应该共有3个骨头。


        图1.从小鼠胫骨,腓骨和股骨中分离骨髓。对小鼠实施安乐死。 A.图像说明切割和移除腿部的位置。 B.用于分离骨髓的小鼠腿部切割位置的示意图。在第1位切割腿(红色虚线)。然后从腿上去除毛发,皮肤和肌肉。然后在红色虚线处切割骨骼,按照指示的顺序隔离骨骼(编号2,3,4和5)。请注意,每个骨骼都在内侧和远侧切割。

      8. 用巨噬细胞分化培养基(MDM)填充26 G针头/ 1 ml注射器(参见配方)。将针尖放入骨中,从骨的两端冲洗骨髓(图2)。这样做是为了股骨,腓骨和胫骨。重复从骨头中冲出骨髓,直到骨头变白。


        图2.冲洗骨骼。 :一种。图像显示骨骼中的骨髓隔室。 B和C.股骨前(B)潮红和(C)潮红(见视频1)。

        视频1.冲洗骨骼以去除骨髓

      9. 收集MDM中的细胞并将其通过70μm细胞过滤器进入无菌锥形管。这样可以去除可能污染细胞的碎片(参见视频2)。

        视频2.清除隔离细胞中的碎片

      10. 在1,950 x g 下将细胞在4℃下旋转5分钟。计数细胞并用巨噬细胞分化培养基(MDM)重悬沉淀,以便可以将5×10 6个细胞和MDM接种到6孔板的每个孔中。
      11. 将板在37℃和5%CO 2 下孵育7天。使用新制造的媒体每三天更换一次媒体。
    2. 巨噬细胞分化为M1或M2
      当BMDM几乎汇合时(第7天),用新的MDM替换。为了使巨噬细胞极化,我们使用脂多糖(LPS)或白细胞介素-4(IL-4)。
      1. 对于经典的M1激活:以100ng / ml的剂量添加脂多糖(LPS)(10μl储备溶液到4ml MDM中)并孵育4小时。
      2. 对于经典的M2激活:将40ng / ml剂量的白细胞介素-4(IL-4)(1.6μl储备溶液加入4ml MDM)中孵育4小时。
    3. 使用U- 13 C葡萄糖进行代谢研究
      1. 用不含葡萄糖,10%活性炭剥离的FBS,100 ng / ml LPS,5 mM U- 13 的培养基替换分化的巨噬细胞(已经用LPS或IL-4处理)中的MDM C葡萄糖(富含99.98%)并在37℃下孵育过夜。我们这样做是因为被调查的基因 Pck1 在禁食状态下被激活,因此我们去除了葡萄糖和胰岛素。&nbsp;
      2. 第二天,收集媒体和细胞。要收集媒体,请移走一半的媒体。保留1毫升培养基用于测量同位素富集。为了收集细胞,刮去孔以去除细胞。吸取细胞和剩余的培养基并放入锥形管中。在1,950 x g 下将细胞在4℃下旋转5分钟,并将沉淀重悬于50μl的1x PBS中。通过Bradford测定法节省20μl用于测量蛋白质浓度。在-20°C冷冻样品以裂解细胞。蛋白质浓度用于代谢物分析中的标准化。细胞用于通过GC-MS分析。在我们的案例中,我们将细胞送到凯斯西储大学小鼠代谢表型中心(MMPC)进行GC-MS分析。
      3. Bradford检测:根据制造商的指示进行检测(快速入门 TM Bradford蛋白质分析)。我们使用2mg / ml BSA作为标准。移取20μl重悬的细胞到1.5ml Eppendorf管中,并从Quick Start TM Bradford Protein Assay中加入1ml 1x Dye试剂。混合样品并在室温下孵育5分钟。将样品放入比色杯中。将分光光度计设置为595 nm。用空白样品(100μl水和1ml 1x染料试剂)将仪器归零。测量BSA标准品和未知(巨噬细胞)的吸光度。从标准曲线确定样品中蛋白质的量(如果2μl样品的等分试样产生相当于250μg/ ml蛋白质的595nm值,则细胞样品将具有2.5mg / ml的蛋白质浓度)。
      4. 对于Pck1 MC-KO 巨噬细胞的研究,将细胞送至Case Western Reserve大学小鼠代谢表型中心(MMPC)进行GC-MS分析。以下部分显示了LPS激活的巨噬细胞中代谢物研究的实例。该实施例研究的目的是比较LPS处理24小时后Pck1 MC-KO (实验)和Pck1 fl / fl (对照)巨噬细胞中的代谢物。

  2. 使用气相色谱质谱(GC-MS)分析柠檬酸循环(CAC)和相关中间体(如柠檬酸盐,琥珀酸盐和苹果酸盐)
    1. 细胞样品制备
      简而言之,这种方法利用甲硅烷基化试剂与醇,酸和胺的快速反应形成甲硅烷基衍生物(Yang et al。,2008a and 2008b; Kombu et al。, 2011; Zhang et al。,2015)。商业甲硅烷基化试剂可以组合使用以加速反应,以及与受阻基团反应。例如,N,O-双(三甲基甲硅烷基)三氟乙酰胺(BSTFA)和三甲基氯硅烷(TMCS)的混合物形成TMS衍生物,或N-甲基-N-(叔丁基二甲基甲硅烷基) - 三氟乙酰胺(MTBSTFA)的混合物和可以使用叔丁基二甲基氯硅烷(TBDMS)形成TBDMS衍生物(Yang et al。,2008a and 2008b; Kombu et al。,2011; Zhang et al 。,2015)
      1. 对于分析物的绝对定量,如前所述(Ko et al。,2018):将50μl细胞加入选定的内部参考标准品,例如~5 nmole的每种代谢物[ 13 < / sup> C 6 ]柠檬酸盐,[ 13 C 4 ]琥珀酸盐,和/或[ 2 H <子> 4 ] - 3-羟基戊二酸。
        注意:内部参考标准的选择取决于所使用的前体示踪剂,使得示踪剂的M +标记与添加的标记标准没有干扰;最好使用非内源性参考标准,如三卡巴酸。&nbsp;
      2. 用3-5ml 5%乙酸的甲醇/水(1:1)提取缓冲液(在冰上冷却)在冰浴上均化细胞2分钟。
      3. 将匀浆在670 x g 下在4℃下离心30分钟。将上清液倒入玻璃试管中并保存在冰上并立即处理
        注意:可将上清液在-80°C冷冻,直至测定GC-MS。
      4. TMSC / TBDMS衍生化程序:将步骤B1b和B1c中收集的100-200μl上清液移液到16mm×125mm一次性硼硅酸盐玻璃管中。
      5. 在氮气下完全干燥(图3)。


        图3.氮气干燥器

      6. 通过添加70μl衍生化TBDMS试剂(在吡啶基溶剂中)并在加热块上在80°C加热1小时来反应(图4)。
        注意:可能在室温下反应过夜。


        图4.带有衍生化管的加热块

      7. 转移到GC插入物和盖子(图5);按照概述(Yang et al。,2008a和2008b; Kombu et al。 ,2011; Zhang et al。,2015)。


        图5.放入GC管中的样品

    2. GC-MS分析
      1. 使用配备Agilent 6890 GC系统的Agilent 5973-MSD和DB-17MS毛细管柱(30 m x 0.25 mm x0.25μm;可使用60 m)分析TMSC或TBDMS衍生物。质谱仪在电子碰撞模式(EI)或氨化学电离模式(CI)和每种分析物的选择性离子监测(SIM) m / z 下操作;当应用稳定同位素时,监测M0-M + m / z 的SIM(Yang et al。,2008a和2008b; Kombu et al。,2011; Zhang et al。,2015)(图6)。


        图6.本协议中使用的GC-MS设备

      2. GC-MS运行后,计算曲线下琥珀酸盐,柠檬酸盐和苹果酸盐的面积以及与内标的比率(表1)。在背景校正后,使用以下公式计算MPE%:
        MPE%= [( 13 C标记为M1至M +,对于每种CAC中间体)x( 13 C标记+未标记) - 1]×100(表1)

        表1.从 13 C-葡萄糖到柠檬酸盐,琥珀酸盐和苹果酸盐的富集摩尔百分比的计算。 M0,M1到M3是根据标记的细胞内代谢物的比例计算的未标记的并且参照提取的细胞的细胞内体积进行校正(Feldberg et al。,2009)。 %MPE计算为%m2 = [( 13 C标记为M1至M +,对于每种CAC中间体)x( 13 C标记+未标记) - 1] x 100.

数据分析

  1. 对于实验设计,对每个样品进行一式三份的稳定同位素研究。一式三份被认为是n = 1.所有实验总共进行n = 6次。
  2. 基于标记的细胞内代谢物与未标记的比例计算M1至M3的MPE,并参照提取的细胞的细胞内体积进行校正(Feldberg 等人,2009)。 %MPE计算为MPE%= [( 13 C标记为M1至M +,对于每种CAC中间体)x( 13 C标记+未标记)]×100。
  3. 对于统计分析,我们使用GraphPad Prism。为了比较两个数据集(Pck1 flox / flox 和Pck1 MC-KO 小鼠),我们进行了学生 t - 测试(图7) 。


    图7.葡萄糖对LPS活化巨噬细胞中枢代谢的贡献。从Pck1 flox / flox 和Pck1 MC-KO 小鼠分离的BMDMs在LPS存在下与[U- 13 C]葡萄糖一起温育16小时,并进行GC-MS计算 13 C对M2的贡献-CAC。值代表每组n = 6的平均值±SEM。 * P &lt;与Pck1 flox / flox 组相比为0.05。

笔记

  1. 由于每次运行的变化,内部标准是必要的。对于该实验,样品(每组n = 6)全部同时在GC-MS上运行以减少变化。
  2. 每只动物一次一个地进行BMDM的分离。例如,对一只Pck1 flox / flox 小鼠实施安乐死,解剖腿并从股骨胫骨和腓骨冲洗骨髓。将细胞保持在冰上冷。然后对下一只动物实施安乐死,依此类推。将细胞保持在冰上直至收集到所有骨髓。
  3. 在电镀BMDM之前预热了培养基。
  4. 为了研究需要快速更新的信号通路(例如,M1激活中的TLR4信号传导或M2激活中的IL-4R信号传导)的启动,建议用更高剂量的刺激物处理细胞(例如,100ng / ml LPS或20ng / ml IL-4)在30分钟内;使用同位素确定代谢途径(例如,柠檬酸循环和相关中间体),用较低剂量的刺激物孵育16-24小时(例如,20 ng / ml LPS建议使用10 ng / ml IL-4)。
  5. 巨噬细胞在激活期间经历形态学变化。与具有圆形形状的未刺激的巨噬细胞相比,LPS处理的巨噬细胞在刺激24小时内显示出类似煎饼的形状。另一方面,IL-4处理的细胞促进细胞伸长。
  6. 从骨髓中分离的细胞将在24小时-48小时内粘附到组织培养板上。 MDM含有M-CSF,可使骨髓细胞分化为巨噬细胞。如果细胞继续漂浮在培养基中,可能会污染细胞。如果发生这种情况,您将需要新鲜培养基并重新分离骨髓细胞。

食谱

  1. 产生巨噬细胞分化培养基(MDM)
    将500毫升无菌Dulbecco's改良Eagle培养基(DMEM)与:
    混合 将55ml 10%胎牛血清(FBS)加入DMEM中 5毫升1x谷氨酰胺到DMEM
    5毫升1x青霉素 - 链霉素(10,000 U / ml Pen,10 mg / ml Strep)
    将5μg巨噬细胞集落刺激因子(M-CSF)加入500ml DMEM中
  2. 5%乙酸的甲醇/水(1:1)萃取缓冲液(在冰上冷却)
    加入50毫升甲醇到50毫升dH 2 O制成甲醇/水,制成100毫升
    在单独的量筒中,将5ml乙酸与95ml上述制备的甲醇/水混合
  3. LPS库存解决方案
    在无菌水和等分试样中补充至40μg/μl
    储存在-80°C
  4. IL-4储备液
    在无菌水和等分试样中补足至100μg/μl
    储存在-80°C

致谢

案例MMPC得到NIH资助U24 DK76174的支持。关于Pck1 MC-KO 小鼠的完整研究已经发表(Ko et al。,2018)。

利益争夺

作者声明他们与本文的内容没有利益冲突。

伦理

所有动物实验均由凯斯西储大学机构动物护理和使用委员会(IACUC)批准。

参考

  1. Feldberg,L.,Venger,I.,Malitsky,S.,Rogachev,I。和Aharoni,A。(2009)。 用于代谢组分析的代谢物的双重标记(DLEMMA):用于鉴定和相对定量的新方法通过双同位素标记和液相色谱 - 质谱分析代谢物。 Anal Chem 81(22):9257-9266。
  2. Ko,C.W.,Counihan,D.,Wu,J.,Hatzoglou,M.,Puchowicz,M.A。和Croniger,C.M。(2018)。 删除 phosphoenolpyruvate carboxykinase 1(Pck1)基因的巨噬细胞更具促炎性的表型。 J Biol Chem 293(9):3399-3409。
  3. Kombu,R。S.,Brunengraber。 H.和Puchowicz,M。A.(2011)。 使用气相色谱 - 质谱法分析柠檬酸循环中间体。在:Metz ,TO(Ed。) Metabolic Profiling:Methods and Protocols。 Humana Press 147-157。
  4. Yang,L.,Kasumov,T.,Kombu,R。S.,Zhu,S.H.,Cendrowski,A.V.,David,F.,Anderson,V.E.,Kelleher,J.K。和Brunengraber,H。(2008a)。 肝脏糖异生和柠檬酸循环的代谢组学和质量同位素分析:II。代谢物标记模式的异质性。 J Biol Chem 283(32):21988-21996。
  5. Yang,L.,Kombu,R。S.,Kasumov,T.,Zhu,S.H.,Cendrowski,A.V.,David,F.,Anderson,V.E.,Kelleher,J.K。和Brunengraber,H。(2008b)。 肝脏糖异生和柠檬酸循环的代谢组学和质量同位素分析。 I.糖异生与卡波氏病之间的相互关系;由氨基氧基乙酸酯和酮酸形成甲氧肟酸酯。 J Biol Chem 283(32):21978-21987。
  6. Zhang,Y.,Zhang,S.,Marin-Valencia,I。和Puchowicz,M。A.(2015)。 饮食诱导的酮症大鼠脑中葡萄糖向氧化代谢的碳分流减少。 J Neurochem 132(3):301-312。
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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. Ko, C. W., Counihan, D., DeSantis, D., Sedor-Schiffhauer, Z., Puchowicz, M. and Croniger, C. M. (2018). Using Stable Isotopes in Bone Marrow Derived Macrophage to Analyze Metabolism. Bio-protocol 8(17): e3003. DOI: 10.21769/BioProtoc.3003.
  2. Ko, C. W., Counihan, D., Wu, J., Hatzoglou, M., Puchowicz, M. A. and Croniger, C. M. (2018). Macrophages with a deletion of the phosphoenolpyruvate carboxykinase 1 (Pck1) gene have a more proinflammatory phenotype. J Biol Chem 293(9): 3399-3409.
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