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Determination of the Glycolysis and Lipogenesis in Culture of Hepatocytes
肝细胞糖酵解和脂肪生成   

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Abstract

Metabolic flux analyses are needed to provide insights into metabolic regulation that occurs in cells. The current protocol describes fast and reproducible methods for determining glycolysis and de novo lipogenesis of hepatocytes. Primary culture of hepatocytes is an ‘in vitro’ model useful to study liver glucose and lipid metabolism (Denechaud et al., 2016). The protocol is divided in 2 parts. Part I: Glycolysis experiment is assessed using the Seahorse extracellular flux (XF) analyser. Glycolysis is determined via the measurement of the extracellular acidification rate (ECAR) of the media, which come predominately from the cellular excretion of lactic acid after the conversion of glucose in pyruvate. Part II: De novo lipogenesis experiment determines the radioactive C14 incorporation in triglycerides (TG) from acetate C14 precursor. After 2 h acetate supplementation to the media lipids are extracted and separated by TLC (Thin Layer Chromatography) prior quantification of newly synthetized TG labelled.

Background

There are different approaches for evaluating glucose and lipid metabolism: metabolite quantification, enzyme activity, and metabolomics... Our protocols focus on metabolic flux analyses of live cells and do not need a metabolomic facility. The Seahorse extracellular flux (XF) analyzer, which is now present in a lot of institution, is a powerful tool for measuring indirectly glycolysis in live cells by determining media pH. Lipogenesis protocol does not need a big investment and is highly reproducible. It could also be determined using tritiated water, which is incorporated by the Fatty Acid Synthase in de novo synthetized lipids.

Part I. Analysis of glycolysis

Materials and Reagents

  1. 60 mm tissue culture dishes (TPP, catalog number: 93060 )
  2. XF assay 24 well cartridge and cell culture microplate V7 (Agilent Technologies, Seahorse Bioscience, catalog number: 100850-001 )
  3. 8-15 weeks old C57BL/6 mouse (Janvier lab)
  4. Collagen type I, rat tail (EMD Millipore, catalog number: 08-115 )
  5. M199 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 41150020 )
  6. Trypsin (2.5%), no phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 15090046 )
  7. Fetal bovine serum (FBS)
  8. DMEM without glucose, L-glutamine, phenol red, sodium pyruvate and sodium bicarbonate, powder, suitable for cell culture (Sigma-Aldrich, catalog number: D5030-10X1L )
  9. D-(+)-glucose solution (Sigma-Aldrich, catalog number: G8769 )
  10. L-glutamine, 200 mM (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  11. XF calibrant solution (Agilent Technologies, Seahorse Bioscience, catalog number: 100840-000 )
  12. PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23225 )
  13. Sodium chloride (NaCl) (AppliChem, catalog number: A3597 )
  14. Phenol red sodium salt (Sigma-Aldrich, catalog number: P5530 )
  15. Tris, pH 7.5 (Applichem, catalog number: A1379 )
  16. EDTA (Sigma-Aldrich, catalog number: E6758 )
  17. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  18. Protease inhibitor (Sigma-Aldrich, catalog number: P8340 )
  19. Glycolysis media (see Recipes)
  20. 9x glucose solution (see Recipes)
  21. Lysis Buffer (see Recipes)

Equipment

  1. CO2-free incubator set to 37 °C (Memmert or other suppliers)
  2. 5% CO2 incubator set to 37 °C (SalvisLab or other suppliers)
  3. Centrifuge
  4. XF 24 extracellular flux analyser (Seahorse Bioscience)
  5. Cell culture hood (Vitaris or other suppliers)
  6. pH meter (Mettler Toledo or other suppliers)
  7. Cell counter (Thermo Fisher Scientific or other suppliers)

Software

  1. Seahorse software (XFReader 24 version 1.6 supply with the XF 24 extracellular flux analyser)

Procedure

  1. Isolate hepatocytes from 8-15 week-old C57BL6/J mice according to Dentin et al. (2004). Dilute at 50 µg/ml the stock collagen I in PBS (do not re-use the solution because collagen I will precipitate at neutral pH). Coat 60 mm culture dish with collagen I (10-20 min at room temperature [RT] are sufficient). Then plate 2 million hepatocytes per dish and incubate in 5% CO2 incubator at 37 °C. 2 h later, change the medium with fresh M199.
  2. At least 6 h before beginning the glycolysis experiment soak the cartridge in a CO2-free incubator at 37 °C with 1 ml of XF calibrant per well.
  3. 24 h after hepatocytes isolation, coat the 24 well of Seahorse XF 24 plate with collagen I (50 µg/ml, 10 min at RT). Then detach the cells by incubating with trypsin for 1-2 min. Stop trypsin with 10% FBS supplemented media. Centrifuge and count the hepatocytes with automatic cell counter. Plate 40,000 cells per well in 100 µl of glycolysis media. Keep 4 wells with only 100 µl of media (without cells) for the background correction of XF24 extracellular flux analyser.
    Note: After seeding, hepatocytes viability should be more than 80%.
  4. Incubate cells 30 min at 37 °C in CO2-free incubator as indicated by the manufacturer (CO2 could participate to the acidification of the media by its hydration to carbonic acid and bicarbonate). Then add 500 µl of 37 °C glycolysis media in each well (volume total of 600 µl).
  5. During these 30 min, load the port A of the cartridge (Figure 1) containing the probes with 75 µl of 9x glucose solution. Prepare the protocol on the Seahorse software according to manufacturer. Choose 3 min mix, 2 min wait and 3 min measurement for the parameters of the Seahorse assay.


    Figure 1. Injection port layout

  6. Start the protocol and load the cartridge with the calibrant to the XF24 Seahorse apparatus (follow the software indications). At the end of the calibration, replace the calibrant plate by the plate with the cells.
  7. After the first measurement, in function of hepatocytes respiration, modify the program: if OCR (oxygen consumption rate) is higher than 200 pMoles oxygen/min, change the program to 4 min mix, 2 min wait and 2 min measurement (according to manufacturer instructions).
    Note: For hepatocytes respiration, OCR should not be below 100 pMoles oxygen/min.
  8. 2 or 3 measurements under basal and 10 measurements after glucose injection are recommended.
  9. At the end of the run, lyse the cells in 40 µl of lysis buffer on ice for 30 min. Quantify the protein level using BCA protein assay.  

Data analysis

  1. The results are normalized by the protein content using the Seahorse software according to the manufacturer and can be represented as Figure 2.
  2. Experiment must be done at least 2 times with 8 replicates.
     

    Figure 2. Representative ECAR plot with injection of glucose. ECAR of C57Bl6/J hepatocytes before and after glucose injection using Seahorse analyser (2 independent experiments, each with 8 replicates).

Notes

  1. The protocol can be adapted to specific needs (use of genetically modified hepatocytes, other cell types).

Recipes

  1. Glycolysis media
    DMEM without glucose, L-glutamine, phenol red, sodium pyruvate and sodium bicarbonate, powder, suitable for cell culture
    143 mM NaCl
    3 mg/L phenol red
    2 mM glutamine
    Adjust pH to 7.35 at 37 °C
  2. 9x glucose solution (225 mM)
    4.55 ml glycolysis media
    450 µl 2.5 M glucose
    Adjust pH to 7.35 at 37 °C
  3. Lysis buffer
    150 mM NaCl
    50 mM Tris (pH 7.5)
    5 mM EDTA
    1% Triton X-100
    Protease inhibitor (1x)

Part II. Analysis of lipogenesis

Materials and Reagents

  1. 6 wells plate (Corning, Costar®, catalog number: 3335 )
  2. Scrapers (Corning, catalog number: 3010 )
  3. Glass Pasteur pipet
  4. Plastic bag
  5. 8-15 weeks old C57BL/6 mouse (Janvier lab)
  6. Nitrogen gas (Carbagaz or other suppliers)
  7. Collagen type I, rat tail (EMD Millipore, catalog number: 08-115 )
  8. M199 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 41150020 )
  9. Dexamethasone (Sigma-Aldrich, catalog number: D1756 )
  10. Insulin (Actrapid HM, Novo Nordisk)
  11. Phosphate-buffered saline (PBS)
  12. D-(+)-glucose solution (Sigma-Aldrich, catalog number: G8769 )
  13. Acetic acid, sodium salt, [1-14C], 1 µCi/µl (PerkinElmer, catalog number: NEC084H001MC )
  14. Chloroform (Sigma-Aldrich, catalog number: 34854 )
  15. Methanol (Sigma-Aldrich, catalog number: 34860 )
  16. Pre-coated TLC-sheets, 0.2 mm silica gel (MACHEREY-NAGEL, catalog number: 805013 )
  17. Iodine (Sigma-Aldrich, catalog number: 207772 )
  18. Diethyl ether (Sigma-Aldrich, catalog number: 309966 )
  19. Petroleum ether (Sigma-Aldrich, catalog number: 320447 )
  20. Acetic acid (Sigma-Aldrich, catalog number: 695092 )
  21. Chloroform/methanol (2:1) (see Recipes)
  22. TLC migration solvent (see Recipes)

Equipment

  1. 5% CO2 incubator set to 37 °C (SalvisLab or other suppliers)
  2. Cell culture hood (Vitaris or other suppliers)
  3. Sonication water bath (Bioruptor)
  4. Vortex (Scientific Industries or other suppliers)
  5. TLC developing chamber for 20 x 20 cm plates (with lid) (CAMAG, catalog number: 022.5250 )
  6. Chemical hood
  7. Cyclone Plus Storage Phosphor Scanner (PerkinElmer, catalog number: C431200 )
  8. MS Multisensitive Phosphor Screens (PerkinElmer, catalog number: 7001723 )
  9. Centrifuge

Software

  1. OptiQuant software (supply with the Cyclone Plus Storage Phosphor Scanner)

Procedure

  1. Isolate hepatocytes from 8-15 week-old C57BL6/J mice according to Dentin et al. (2004). As previously described, coat the wells of a 6 wells-plate with collagen I (10 min at RT) and plate 1 million cells per well. After 2 h, change the medium with fresh M199.
  2. The next day, treat the cells with the non-stimulated (G5) and stimulated (G25i) conditions for 24 h:
    a. Low-glucose (G5): M199 media (glucose 5 mM) plus dexamethasone 10-7 M.
    b. High-glucose insulin (G25i): M199 media (glucose 5 mM) complemented to 25 mM glucose, plus insulin 100 nM and dexamethasone 10-7 M.
  3. Add acetate C14 (0.5 µCi per well [0.5 µl]) in the media. C14 will be incorporated in de novo synthetized lipids.
  4. After 2 h, wash the cells 2 times with PBS and scrap the cells of each well in 400 µl of PBS. Then the 400 µl is divided into 100 µl and 300 µl for protein quantification and lipid extraction respectively as described below.
  5. For protein quantification. Freeze at -20 °C to improve the lysis of the protein. (Optional: sonicate 15 sec in sonication water bath). Quantify using BCA protein assay.
  6. From the 300 µl left, perform lipid extraction under a chemical hood as follow. Add 100 µl of M199 media with phenol red to facilitate the recognition of the 2 phases in the later stage. Then add 1.6 ml of chloroform/methanol (2:1) and vortex 5 to 10 min. Centrifuge 5 min at 15,000 x g (RCF) at RT and collect the organic phase (lower phase) with glass Pasteur pipet. The phenol red helps to distinguish two phases. Then evaporate under nitrogen blowing (approx. 20 to 40 min) and resuspend in 150 µl of chloroform/methanol (2:1).
  7. Add 1 cm of TLC migration solvent in the TLC developing chamber 20 min before loading the TLC plate in the TLC developing chamber.
  8. Mark a line with a pencil at 2 cm from the bottom of the TLC plate. Samples will be loaded every 1.5-2 cm on this line. Load 40 µl as a dot on the TLC plate. To avoid too large dots, at the same time you load, blow nitrogen for immediate drying.
  9. Load the TLC plate in the TLC developing chamber. Allow TLC plate migration to 1 cm of the top (approx. 45 min).
  10. (Optional) After TLC migration, the lipid can be visualised. Under the chemical hood, dry the TLC plate under the hood and colour lipid with iodine in a clean TLC chamber. Add some iodine stones in the bottom of the TLC chamber and place the TLC plate. Wait until the brown colour appears (10 to 20 min). You can take a picture under the hood or you can put the plate in a closed plastic bag for scanning. Recover the iodine stones in a close bottle (they can be used indefinitely). Note that the coloration disappears outside of the chamber. Remove the coloration under the hood (approx. 10 min).
  11. Expose the TLC to the MS Multisensitive Phosphor Screens for at least 5 days in a film cassette. Then scan the screen using Cyclone Plus Storage Phosphor Scanner (Figure 3).


    Figure 3. Representative scan after migration of the sample on TLC plate. Visualization of C14 labelled lipids after TLC migration. Samples from hepatocytes treated 24 h with G5 or G25i where load on the bottom of the plate. TGs are highlighted.

Data analysis

  1. Using the OptiQuant software supplied with the Scanner, determine the levels of incorporated C14 in the TG by quantifying the TG spot intensity.
  2. The results are normalized by the protein content and expressed as relative values respect to the control condition (G25i) taken as 100% of incorporation.
  3. Experiments must be performed 3 times in triplicate, as minimum.

Notes

  1. Each condition has to be performed in triplicate. G5 and G25i are conditions were lipogenesis is low and high respectively in wild type hepatocytes. Hepatocytes in G25i are expected to have 3 times more lipogenesis (Denechaud et al., 2016).
  2. Lipid extraction, TLC migration, TLC coloration (iodine) have to be performed under a chemical hood.
  3. Any phosphorimager scanner can be used for scanning the TLC plate.
  4. The protocol can be adapted to specific needs (specific culture conditions, use of genetically modified hepatocytes, other cell types).

Recipes

  1. Chloroform/methanol (2:1)
    2 volumes of chloroform for 1 volume of methanol
  2. TLC migration solvent
    127 ml petroleum ether
    22 ml diethyl ether
    0.5 ml acetic acid

Acknowledgments

We thank Dr. Isalel Lopez-Mejia for helpful discussions and for her expertise in XF24 extracellular flux analyser. This work was supported by grants from the Swiss Ligue Contre le Cancer, the Swiss National Science Foundation and the Fondation de France. We thank Dr. K. Schooonjans (EPFL, Lausanne, Switzerland) for his previous work (Oosterveer et al., 2012) that we adapt and modify the glycolysis protocol.

References

  1. Dentin, R., Pegorier, J. P., Benhamed, F., Foufelle, F., Ferre, P., Fauveau, V., Magnuson, M. A., Girard, J. and Postic, C. (2004). Hepatic glucokinase is required for the synergistic action of ChREBP and SREBP-1c on glycolytic and lipogenic gene expression. J Biol Chem 279(19): 20314-20326.
  2. Denechaud, P. D., Lopez-Mejia, I. C., Giralt, A., Lai, Q., Blanchet, E., Delacuisine, B., Nicolay, B. N., Dyson, N. J., Bonner, C., Pattou, F., Annicotte, J. S. and Fajas, L. (2016). E2F1 mediates sustained lipogenesis and contributes to hepatic steatosis. J Clin Invest 126(1): 137-150.
  3. Oosterveer, M. H., Mataki, C., Yamamoto, H., Harach, T., Moullan, N., van Dijk, T. H., Ayuso, E., Bosch, F., Postic, C., Groen, A. K., Auwerx, J. and Schoonjans, K. (2012). LRH-1-dependent glucose sensing determines intermediary metabolism in liver. J Clin Invest 122(8): 2817-2826.

简介

需要代谢通量分析来提供细胞中发生的代谢调节的见解。目前的协议描述了确定糖酵解和肝细胞脂肪生成的快速和可重复的方法。肝细胞的原代培养是用于研究肝葡萄糖和脂质代谢的"体外"模型(Denechaud等人,2016)。协议分为2部分。第I部分:使用海马细胞外通量(XF)分析仪评估糖酵解实验。糖酵解通过测量培养基的细胞外酸化速率(ECAR)来确定,所述培养基主要来自丙酮酸中葡萄糖转化后乳酸的细胞排泄。第II部分:新生脂肪生成实验测定来自乙酸盐C 14+前体的甘油三酯(TG)中的放射性C 14+掺入。在2小时后,向培养基中补加乙酸盐,提取脂质,并在新合成的TG标记的定量之前通过TLC(薄层色谱)分离。

[背景] 有不同的方法来评估葡萄糖和脂质代谢:代谢物定量,酶活性和代谢组学...我们的协议聚焦于活细胞的代谢通量分析并且不需要代谢组学设施。海马细胞外通量(XF)分析仪,现在存在于很多机构,是通过确定培养基pH值间接测量活细胞间接糖酵解的强大工具。脂肪生成协议不需要大投资,是高度可重复的。也可以使用氚化水确定,其通过脂肪酸合酶引入新鲜合成的脂质中。

第一部分糖酵解分析

材料和试剂

  1. 60mm组织培养皿(TPP,目录号:93060)
  2. XF测定24孔盒和细胞培养微板V7(Agilent Technologies,Seahorse Bioscience,目录号:100850-001)
  3. 8-15周龄C57BL/6小鼠(Janvier实验室)
  4. 胶原I型,大鼠尾(EMD Millipore,目录号:08-115)
  5. M199培养基(Thermo Fisher Scientific,Gibco TM ,目录号:41150020)
  6. 胰蛋白酶(2.5%),无酚红(Thermo Fisher Scientific,Gibco TM ,目录号:15090046)
  7. 胎牛血清(FBS)
  8. 适于细胞培养的DMEM(不含葡萄糖,L-谷氨酰胺,酚红,丙酮酸钠和碳酸氢钠)粉末(Sigma-Aldrich,目录号:D5030-10X1L)
  9. D - (+) - 葡萄糖溶液(Sigma-Aldrich,目录号:G8769)
  10. L-谷氨酰胺,200mM(Thermo Fisher Scientific,Gibco TM ,目录号:25030081)
  11. XF校准溶液(Agilent Technologies,Seahorse Bioscience,目录号:100840-000)
  12. PierceBCA蛋白测定试剂盒(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:23225)
  13. 氯化钠(NaCl)(AppliChem,目录号:A3597)
  14. 酚红钠盐(Sigma-Aldrich,目录号:P5530)
  15. Tris,pH7.5(Applichem,目录号:A1379)
  16. EDTA(Sigma-Aldrich,目录号:E6758)
  17. Triton X-100(Sigma-Aldrich,目录号:X100)
  18. 蛋白酶抑制剂(Sigma-Aldrich,目录号:P8340)
  19. 糖酵解介质(参见配方)
  20. 9x葡萄糖溶液(见配方)
  21. 裂解缓冲液(参见配方)

设备

  1. CO 2 sub-free培养箱设置为37℃(Memmert或其他供应商)
  2. 5%CO 2孵育器设置为37℃(Salvis Lab或其他供应商)
  3. 离心机
  4. XF 24胞外通量分析仪(Seahorse Bioscience)
  5. 细胞培养罩(Vitaris或其他供应商)
  6. pH计(Mettler Toledo或其他供应商)
  7. 细胞计数器(Thermo Fisher Scientific或其他供应商)

软件

  1. 海马软件(XFReader 24版本1.6供应XF 24细胞外通量分析仪)

程序

  1. 根据Dentin等分离来自8-15周龄C57BL6/J小鼠的肝细胞。 (2004)。在PBS中稀释50μg/ml的股票胶原蛋白I(不要重复使用溶液,因为胶原蛋白I将在中性pH下沉淀)。用胶原蛋白I(在室温[RT]下10-20分钟)涂覆60mm培养皿就足够了。然后每板平板200万肝细胞,并在5%CO 2培养箱中在37℃孵育。 2小时后,更换新鲜的M199培养基
  2. 在开始糖酵解实验之前至少6小时将该盒子在37℃的CO 2无培养箱中用每孔1ml XF校准物浸泡。
  3. 在肝细胞分离后24小时,用胶原I(50μg/ml,室温下10分钟)包被Seahorse XF 24板的24孔。然后通过与胰蛋白酶孵育1-2分钟分离细胞。停止胰蛋白酶用10%FBS补充培养基。离心并用自动细胞计数器计数肝细胞。在100μl糖酵解介质中每孔铺板40,000个细胞。保持4个井,只有100微升的媒体(没有细胞)的XF24细胞外通量分析仪的背景校正 注意:播种后,肝细胞活力应大于80%。
  4. 在制造商指示的CO 2无培养箱中在37℃下孵育细胞30分钟(CO 2可以通过其水合成为碳酸来参与培养基的酸化和碳酸氢盐)。然后在每个孔中加入500μl的37℃糖酵解培养基(总体积为600μl)。
  5. 在这30分钟内,加载盒的端口A(图1),其中含有75μl的9x葡萄糖溶液的探针。根据制造商在Seahorse软件上准备协议。选择3分钟混合,2分钟等待和3分钟测量海马测定的参数

    图1.注入端口布局

  6. 启动协议并将校准液加载到XF24海马装置(按照软件指示)。在校准结束时,将校准板用电池板替换。
  7. 在第一次测量后,在肝细胞呼吸的功能中,修改程序:如果OCR(氧消耗速率)高于200pMoles氧/分钟,将程序改变为4分钟混合,2分钟等待和2分钟测量(根据制造商说明)。
    注意:对于肝细胞呼吸,OCR不应低于100 pMoles oxyge/min。
  8. 建议在基础下进行2或3次测量,在葡萄糖注射后进行10次测量
  9. 在运行结束时,在冰上裂解细胞在40μl裂解缓冲液中30分钟。使用BCA蛋白测定法定量蛋白质水平。  

数据分析

  1. 使用Seahorse软件根据制造商通过蛋白质含量将结果标准化,并且可以表示为图2
  2. 实验必须至少进行2次,重复次数为8次。
     

    图2.使用海马分析器(2次独立实验,每次重复8次)在注射葡萄糖之前和之后的C57B16/J肝细胞的ECAR的葡萄糖注射的代表性ECAR图。

笔记

  1. 该方案可以适应具体需要(使用遗传修饰的肝细胞,其他细胞类型)。

食谱

  1. 糖酵解介质
    无葡萄糖,L-谷氨酰胺,酚红,丙酮酸钠和碳酸氢钠的DMEM,粉末,适用于细胞培养
    143 mM NaCl 3 mg/L酚红
    2mM谷氨酰胺 在37℃下将pH调节至7.35
  2. 9x葡萄糖溶液(225mM) 4.55ml糖酵解介质
    450μl2.5M葡萄糖
    在37℃下将pH调节至7.35
  3. 裂解缓冲液
    150mM NaCl 50mM Tris(pH7.5) 5 mM EDTA
    1%Triton X-100 蛋白酶抑制剂(1x)

第II部分。脂肪生成分析

材料和试剂

  1. 6孔板(Corning,Costar ,目录号:3335)
  2. 刮刀(Corning,目录号:3010)
  3. 玻璃巴斯德移液器
  4. 塑料袋
  5. 8-15周龄C57BL/6小鼠(Janvier实验室)
  6. 氮气(Carbagaz或其他供应商)
  7. 胶原I型,大鼠尾(EMD Millipore,目录号:08-115)
  8. M199培养基(Thermo Fisher Scientific,Gibco TM ,目录号:41150020)
  9. 地塞米松(Sigma-Aldrich,目录号:D1756)
  10. 胰岛素(Actrapid HM,Novo Nordisk)
  11. 磷酸盐缓冲盐水(PBS)
  12. D - (+) - 葡萄糖溶液(Sigma-Aldrich,目录号:G8769)
  13. 乙酸,钠盐,[1- 14 C],1μCi/μl(PerkinElmer,目录号:NEC084H001MC)
  14. 氯仿(Sigma-Aldrich,目录号:34854)
  15. 甲醇(Sigma-Aldrich,目录号:34860)
  16. 预涂覆的TLC片,0.2mm硅胶(MACHEREY-NAGEL,目录号:805013)
  17. 碘(Sigma-Aldrich,目录号:207772)
  18. 二乙醚(Sigma-Aldrich,目录号:309966)
  19. 石油醚(Sigma-Aldrich,目录号:320447)
  20. 乙酸(Sigma-Aldrich,目录号:695092)
  21. 氯仿/甲醇(2:1)(参见配方)
  22. TLC迁移溶剂(参见配方)

设备

  1. 5%CO 2孵育器设置为37℃(Salvis Lab或其他供应商)
  2. 细胞培养罩(Vitaris或其他供应商)
  3. 超声水浴(Bioruptor)
  4. Vortex(科学工业或其他供应商)
  5. 用于20×20cm平板(带盖)(CAMAG,目录号:022.5250)的TLC显影室
  6. 化学发动机罩
  7. Cyclone Plus存储荧光粉扫描仪(PerkinElmer,目录号:C431200)
  8. MS多重敏感荧光屏(PerkinElmer,目录号:7001723)
  9. 离心机

软件

  1. OptiQuant软件(配备Cyclone Plus存储荧光扫描仪)

程序

  1. 根据Dentin等分离来自8-15周龄C57BL6/J小鼠的肝细胞。 (2004)。如前所述,用胶原I(室温下10分钟)包被6孔板的孔,每孔涂布1百万个细胞。 2小时后,用新鲜的M199更换培养基
  2. 第二天,用非刺激(G5)和刺激(G25i)条件处理细胞24小时:
    一个。低葡萄糖(G5):M199培养基(葡萄糖5mM)加上地塞米松10μM-7 M.
    b。高葡萄糖胰岛素(G25i):补充有25mM葡萄糖,加胰岛素100nM和地塞米松10μM7的M199培养基(葡萄糖5mM)。
  3. 在培养基中加入乙酸盐14(0.5μCi/孔[0.5μl])。 C 14 将被并入新的合成脂质中。
  4. 2小时后,用PBS洗涤细胞2次,并将每孔的细胞倒入400μlPBS中。然后将400μl分成100μl和300μl,分别用于蛋白质定量和脂质提取,如下所述
  5. 用于蛋白质定量。在-20°C冷冻以改善蛋白质的裂解。 (任选:在超声水浴中超声15秒)。使用BCA蛋白测定法定量
  6. 从300μl左边,在化学罩下进行脂质提取如下。添加100微升的M199介质与酚红,以方便识别后阶段的两个阶段。然后加入1.6ml氯仿/甲醇(2:1)并涡旋5-10分钟。在RT下以15,000×g(RCF)离心5分钟,并用玻璃巴斯德吸管收集有机相(下相)。酚红有助于区分两相。然后在氮气吹扫下蒸发(约20至40分钟),并用150μl氯仿/甲醇(2:1)重悬。
  7. 在TLC显影室中加入1cm的TLC迁移溶剂,在TLC显影室中装入TLC板之前20分钟
  8. 在距离TLC板底部2cm处用铅笔标记线。样品将在每条1.5-2厘米的线上加载。加载40微升作为点在TLC板上。为避免太大的点,在加载的同时,吹氮气即可干燥
  9. 将TLC板装载在TLC显影室中。使TLC板迁移至顶部1cm(约45分钟)
  10. (任选)TLC迁移后,可以显现脂质。在化学罩下,在罩下干燥TLC板并在干净的TLC室中用碘对脂质进行染色。在TLC室的底部加入一些碘石并放置TLC板。等待,直到出现棕色(10到20分钟)。您可以在引擎盖下拍摄照片,或者您可以将板放在封闭的塑料袋中进行扫描。在一个封闭的瓶子中恢复碘结石(它们可以无限期使用)。注意,着色在室外消失。除去罩下的着色(约10分钟)。
  11. 在薄膜盒中将TLC暴露于MS多重敏感荧光屏至少5天。然后使用Cyclone Plus存储荧光扫描仪扫描屏幕(图3)。


    图3.在TLC板上样品迁移后的代表性扫描。在TLC迁移后C 14标记的脂质的可视化。来自肝细胞的样品用G5或G25i处理24小时,其中在板的底部上的负载。突出显示TG。

数据分析

  1. 使用扫描仪附带的OptiQuant软件,通过量化TG光斑强度来确定TG中结合的C 14 的水平。
  2. 结果通过蛋白质含量归一化,并表示为相对于作为100%掺入的对照条件(G25i)的相对值。
  3. 实验必须进行三次,一式三份,最少。

笔记

  1. 每个条件必须一式三份执行。 G5和G25i是在野生型肝细胞中脂肪生成分别低和高的条件。 G25i中的肝细胞预期具有3倍以上的脂肪生成(Denechaud等人,2016)。
  2. 脂质提取,TLC迁移,TLC着色(碘)必须在化学罩下进行
  3. 任何phosphorimager扫描仪都可用于扫描TLC板
  4. 该方案可以适应具体需要(特定培养条件,使用转基因肝细胞,其他细胞类型)。

食谱

  1. 氯仿/甲醇(2:1)
    2体积的氯仿/1体积的甲醇
  2. TLC迁移溶剂
    127ml石油醚
    22毫升乙醚 0.5ml乙酸

致谢

我们感谢Isalel Lopez-Mejia博士有益的讨论和她在XF24细胞外通量分析仪的专业知识。这项工作得到瑞士血液癌症研究所,瑞士国家科学基金会和法国基金会的资助。我们感谢K. Schooonjans博士(EPFL,洛桑,瑞士)的他以前的工作(Oosterveer等人,2012),我们适应和修改糖酵解方案。

参考文献

  1. Dentin,R.,Pegorier,JP,Benhamed,F.,Foufelle,F.,Ferre,P.,Fauveau,V.,Magnuson,MA,Girard,J.and Postic, ChREBP和SREBP-1c对糖酵解的协同作用需要肝葡萄糖激酶和脂质基因表达。 J Biol Chem 279(19):20314-20326。
  2. Denichaud,PD,Lopez-Mejia,IC,Giralt,A.,Lai,Q.,Blanchet,E.,Delacuisine,B.,Nicolay,BN,Dyson,NJ,Bonner,C.,Pattou,F.,Annicotte, JS和Fajas,L.(2016)。  E2F1介导持续的脂肪生成和有助于肝脂肪变性。 J Clin Invest 126(1):137-150。
  3. Oosterveer,MH,Mataki,C.,Yamamoto,H.,Harach,T.,Moullan,N.,van Dijk,TH,Ayuso,E.,Bosch,F.,Postic,C.,Groen,AK,Auwerx, J.和Schoonjans,K.(2012)。  LRH -1依赖性葡萄糖检测决定肝中的中间代谢。 122(8):2817-2826。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Denechaud, P. and Fajas, L. (2016). Determination of the Glycolysis and Lipogenesis in Culture of Hepatocytes. Bio-protocol 6(21): e1993. DOI: 10.21769/BioProtoc.1993.
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