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Metabolic Assays for Detection of Neutral Fat Stores
代谢试验检测中性脂肪储存   

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Abstract

Lipid droplets (LDs) are ubiquitous intracellular structures whose formation, growth, and maintenance are highly regulated (Wang et al., 2013; Ranall et al., 2011; Goodman, 2009). Lipid metabolism and droplet dynamics are of considerable interest to agriculture, biofuel production, viral pathology, nutrition, and cancer biology (Walther and Farese, 2009; Liu et al., 2010). Accumulation of fatty acids and neutral lipids in nonadipose tissues is cytotoxic (Kourtidis et al., 2009). BODIPY 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene) is the standard dye to study LDs within adipocytes. BODIPY 493/503 contains a nonpolar structure that, upon binding to neutral lipid, emits a green fluorescence signal with a narrow wavelength range, making it an ideal fluorophore for multi-labeling experiments. The hydrophobic nature of the dye molecules promotes rapid entry into the nonpolar environment of LDs (Listenberge and Brown, 2007). Gocze and Freeman showed that the lipid fluorescent variability is significantly lower when using BODIPY493/503 compared to Nile Red, suggesting that it may be more specific for the LD (Gocze and Freeman, 1994). Here, we describe a BODIPY 493/503 assay for the detection of neural fat stores in cultured cells (Figure 1) (Wang et al., 2013).



Figure 1. MCF7 cells were treated with 250 μM palmitate or vehicle control for 24 h. A number of breast cancer cells possess a lipogenic metabolic phenotype that makes them especially sensitive to the addition of physiological concentrations of exogenous saturated fatty acids, such as palmitate. Although palmitate supplementation induces cell death in HER2/neu-positive cells, other breast cancer sub-types, including MCF-7 cells, accumulate the fatty acid which leads to significant increases in intracellular triglyceride fat stores. Cells were fixed and stained for fat stores with BODIPY 493/503 (green). Hoechst 33342 (blue) was used for nuclei staining.

Materials and Reagents

  1. Cell line suitable for testing, MCF7 (ATCC, catalog number: HTB-22 TM) cells perform well as a staining control
  2. Dulbecco’s Modified Eagle’s Medium (DMEM) (high glucose with L-glutamine) (Thermo Fisher Scientific, catalog number: SH3024301 ) or other media appropriate for MCF7 cell culture
  3. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F4135 )
  4. Phosphate buffered saline (PBS) (e.g. HyClone, catalog number: SH30258-02 ) or Dulbecco's phosphate buffered saline (with Ca2+ and Mg2+) (DPBS) (e.g. Sigma-Aldrich, catalog number: D1283 )
  5. Sodium palmitate, (used as positive control) (Sigma-Aldrich, catalog number: P9767 )
  6. Dimethyl sulfoxide (Sigma-Aldrich, catalog number: D8418 )
  7. BODIPY 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene) (Life Technologies, catalog number: D-3922 )
  8. 37% formaldehyde (Thermo Fisher Scientific, catalog number: F79 )
  9. Hoechst 33342 (Life Technologies, catalog number: H21492 )
  10. 500x BODIPY 493/503 stock solution (see Recipes)
  11. 10,000x Hoechst 33342 stock solution (see Recipes)

Equipment

  1. 96-well tissue culture plate suitable for imaging, (e.g. Corning, Costar®, catalog number: 3603 )
  2. Cell culture incubator at 37 °C with 5% CO2
  3. Fluorescence microscope or automated imaging system (e.g. IN Cell Analyzer, GE Healthcare)

Procedure

  1. Plate 10,000-20,000 cells per well in 100 μl DMEM high glucose with L-glutamine, 10% FBS, in the 96-well plate. Doing a serial dilution can be helpful to determine the effect of cell number on BODIPY intensity.
  2. Incubate the cells at 37 °C overnight.
  3. Dilute the sodium palmitate stock solution in pre warmed DMEM high glucose with L-glutamine, 10% FBS. A range of 50-500 μM is useful to demonstrate BODIPY-based lipid content relative quantification. Prepare appropriate vehicle controls.
  4. Carefully aspirate medium from the 96-well plate and replace with treatment medium (use 100 μl per well).
  5. Incubate the cells at 37 °C overnight.
  6. Prepare a 5% formaldehyde solution in PBS. Add 100 μl per well directly into the tissue culture medium to achieve a final formaldehyde concentration of 2.5%.
  7. Incubate for 15 min at room temperature.
  8. Prepare the staining solution; dilute the BODIPY and Hoechst stock solutions in PBS to a working concentration of 10 μg/ml BODIPY and 1 μg/ml Hoechst. Calculate 100 μl per well. Both dyes can be added in parallel, there is no need for sequential staining.
  9. Carefully remove the formaldehyde from the cells and wash twice with 100 μl of PBS per well.
  10. Add 100 μl staining solution per well.
  11. Incubate for 30 min at room temperature, in the dark.
  12. Wash twice with 100 μl of PBS per well.
  13. Remove PBS, add 130 μl of PBS per well (only required for IN Cell Analyzer).
  14. BODIPY and nuclei Hoechst fluorescence are imaged and quantified using fluorescence microscopy with appropriate filter sets. Our laboratory makes extensive use of the INCell Analyzer 2200 and INCell Investigator software for these measurements. Fluorescence intensity per cell is proportional to the neutral lipid content in the cell.

Notes

  1. Solubilize sodium palmitate in DMSO at 75 mM immediately before usage, do not store.
  2. If many cells are lost from the plate during washing with regular PBS, use DPBS for washing and preparing working solutions of BODIPY and Hoechst.
  3. Care should be taken to stain at approximately equal cell densities when comparing different cells or culture conditions.

Recipes

  1. 500x BODIPY 493/503 stock solution
    Solubilize BODIPY 493/503 in DMSO at 5 mg/ml
    Aliquot and stored at -20 °C in the dark
  2. 10,000x Hoechst 33342 stock solution
    Solubilize Hoechst 33342 in DMSO at 10 mg/ml
    Aliquot and stored at -20 °C in the dark

Acknowledgments

Supported by U.S. Army Medical Research Acquisition Activity W8IWXH-04-1-0474 and NCI 1R01CA136658 to DSC.

References

  1. Gocze, P. M. and Freeman, D. A. (1994). Factors underlying the variability of lipid droplet fluorescence in MA-10 Leydig tumor cells. Cytometry 17(2): 151-158.
  2. Goodman, J. M. (2009). Demonstrated and inferred metabolism associated with cytosolic lipid droplets. J Lipid Res 50(11): 2148-2156.
  3. Kourtidis, A., Srinivasaiah, R., Carkner, R. D., Brosnan, M. J. and Conklin, D. S. (2009). Peroxisome proliferator-activated receptor-gamma protects ERBB2-positive breast cancer cells from palmitate toxicity. Breast Cancer Res 11(2): R16.
  4. Listenberger, L. L. and Brown, D. A. (2007). Fluorescent detection of lipid droplets and associated proteins. Curr Protoc Cell Biol Chapter 24: Unit 24 22.
  5. Liu, H., Liu, J. Y., Wu, X. and Zhang, J. T. (2010). Biochemistry, molecular biology, and pharmacology of fatty acid synthase, an emerging therapeutic target and diagnosis/prognosis marker. Int J Biochem Mol Biol 1(1): 69-89.
  6. Ranall, M. V., Gabrielli, B. G. and Gonda, T. J. (2011). High-content imaging of neutral lipid droplets with 1,6-diphenylhexatriene. Biotechniques 51(1): 35-36, 38-42.
  7. Walther, T. C. and Farese, R. V., Jr. (2009). The life of lipid droplets. Biochim Biophys Acta 1791(6): 459-466.
  8. Wang, X., Sun, Y., Wong, J. and Conklin, D. S. (2013). PPARgamma maintains ERBB2-positive breast cancer stem cells. Oncogene 32(49): 5512-552.

简介

脂质滴(LD)是普遍存在的细胞内结构,其形成,生长和维持是高度调节的(Wang等人,2013; Ranall等人,2011; Goodman, 2009)。脂质代谢和液滴动力学对农业,生物燃料生产,病毒病理学,营养和癌症生物学是相当感兴趣的(Walther和Farese,2009; Liu等人,2010)。在非脂肪组织中脂肪酸和中性脂质的积累是细胞毒性的(Kourtidis等人,2009)。 BODIPY 493/503(4,4-二氟-1,3,5,7,8-五甲基-4-Bora-​​3a,4a-Diaza-s-Indacene)是研究脂肪细胞内LD的标准染料。 BODIPY 493/503含有非极性结构,在结合中性脂质时,发射具有窄波长范围的绿色荧光信号,使其成为用于多标记实验的理想荧光团。染料分子的疏水性质促进快速进入LD的非极性环境(Listenberge和Brown,2007)。 Gocze和Freeman显示,当使用BODIPY493/503与Nile Red相比时,脂质荧光变异性显着降低,表明其可能对LD更具特异性(Gocze和Freeman,1994)。在这里,我们描述了BODIPY 493/503测定用于检测培养的细胞中的神经脂肪储存(图1)(Wang等人,2013)。



图1. MCF7细胞用250μM棕榈酸盐或载体对照处理24小时。 一些乳腺癌细胞具有脂肪代谢表型,使得它们对添加生理浓度的外源饱和脂肪酸(例如棕榈酸盐)特别敏感。虽然棕榈酸补充诱导HER​​2/neu阳性细胞中的细胞死亡,但其它乳腺癌亚型(包括MCF-7细胞)积聚了导致细胞内甘油三酯脂肪储存显着增加的脂肪酸。将细胞固定并用BODIPY 493/503(绿色)对脂肪储存进行染色。 Hoechst 33342(蓝色)用于细胞核染色。

材料和试剂

  1. 适合测试的细胞系MCF7(ATCC,目录号:HTB-22 TM )作为染色对照表现良好
  2. Dulbecco改良的Eagle培养基(DMEM)(具有L-谷氨酰胺的高葡萄糖)(Thermo Fisher Scientific,目录号:SH3024301)或适用于MCF7细胞培养物的其它培养基
  3. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F4135)
  4. 磷酸盐缓冲盐水(PBS)(例如HyClone,目录号:SH30258-02)或Dulbecco's磷酸盐缓冲盐水(具有Ca 2+和Mg 2+) (DPBS)(例如Sigma-Aldrich,目录号:D1283)。
  5. 棕榈酸钠(用作阳性对照)(Sigma-Aldrich,目录号:P9767)
  6. 二甲基亚砜(Sigma-Aldrich,目录号:D8418)
  7. BODIPY 493/503(4,4-二氟-1,3,5,7,8-五甲基-4-Bora-3a,4a-Diaza-s-Indacene)(Life Technologies,目录号:D-3922) />
  8. 37%甲醛(Thermo Fisher Scientific,目录号:F79)
  9. Hoechst 33342(Life Technologies,目录号:H21492)
  10. 磷酸盐缓冲盐水(PBS)(例如HyClone,目录号:SH30258-02)或Dulbecco's磷酸盐缓冲盐水(具有Ca 2+和Mg 2+) (DPBS)(例如Sigma-Aldrich,目录号:D1283)。
  11. 棕榈酸钠(用作阳性对照)(Sigma-Aldrich,目录号:P9767)
  12. 二甲基亚砜(Sigma-Aldrich,目录号:D8418)
  13. BODIPY 493/503(4,4-二氟-1,3,5,7,8-五甲基-4-Bora-3a,4a-Diaza-s-Indacene)(Life Technologies,目录号:D-3922) />
  14. 37%甲醛(Thermo Fisher Scientific,目录号:F79)
  15. Hoechst 33342(Life Technologies,目录号:H21492)... Fluorescence microscope or automated imaging system (e.g. IN Cell Analyzer, GE Healthcare)

Procedure

  1. Plate 10,000-20,000 cells per well in 100 μl DMEM high glucose with L-glutamine, 10% FBS, in the 96-well plate. Doing a serial dilution can be helpful to determine the effect of cell number on BODIPY intensity.
  2. Incubate the cells at 37 °C overnight.
  3. Dilute the sodium palmitate stock solution in pre warmed DMEM high glucose with L-glutamine, 10% FBS. A range of 50-500 μM is useful to demonstrate BODIPY-based lipid content relative quantification. Prepare appropriate vehicle controls.
  4. Carefully aspirate medium from the 96-well plate and replace with treatment medium (use 100 μl per well).
  5. Incubate the cells at 37 °C overnight.
  6. Prepare a 5% formaldehyde solution in PBS. Add 100 μl per well directly into the tissue culture medium to achieve a final formaldehyde concentration of 2.5%.
  7. Incubate for 15 min at room temperature.
  8. Prepare the staining solution; dilute the BODIPY and Hoechst stock solutions in PBS to a working concentration of 10 μg/ml BODIPY and 1 μg/ml Hoechst. Calculate 100 μl per well. Both dyes can be added in parallel, there is no need for sequential staining.
  9. Carefully remove the formaldehyde from the cells and wash twice with 100 μl of PBS per well.
  10. Add 100 μl staining solution per well.
  11. Incubate for 30 min at room temperature, in the dark.
  12. Wash twice with 100 μl of PBS per well.
  13. Remove PBS, add 130 μl of PBS per well (only required for IN Cell Analyzer).
  14. BODIPY and nuclei Hoechst fluorescence are imaged and quantified using fluorescence microscopy with appropriate filter sets. Our laboratory makes extensive use of the INCell Analyzer 2200 and INCell Investigator software for these measurements. Fluorescence intensity per cell is proportional to the neutral lipid content in the cell.

Notes

  1. 在使用前立即溶解棕榈酸钠在75mM的DMSO中,不储存
  2. 如果在用常规PBS洗涤期间许多细胞从培养板丢失,使用DPBS洗涤并制备BODIPY和Hoechst的工作溶液。
  3. 当比较不同的细胞或培养条件时,应当注意大致相等的细胞密度染色

食谱

  1. 500x BODIPY 493/503储备液
    将BODIPY 493/503在DMSO中以5mg/ml溶解 等分并在-20℃下在黑暗中保存
  2. 10,000x Hoechst 33342储备溶液
    使溶解在DMSO中的Hoechst 33342 10mg/ml,
    等分并在-20℃下在黑暗中保存

致谢

由美国陆军医学研究获得活动W8IWXH-04-1-0474和NCI 1R01CA136658支持到DSC。

参考文献

  1. Gocze,P.M。和Freeman,D.A。(1994)。 MA-10Leydig肿瘤细胞中脂质滴荧光变异性的因素 < em> Cytometry 17(2):151-158。
  2. Goodman,J.M。(2009)。 与细胞溶质脂质液滴相关的已证明和推断的代谢。 J Lipid Res/em> 50(11):2148-2156。
  3. Kourtidis,A.,Srinivasaiah,R.,Carkner,R.D.,Brosnan,M.J.and Conklin,D.S.(2009)。 Peroxisome增殖物激活受体-γ保护ERBB2阳性乳腺癌细胞免受棕榈酸毒性。 Breast Cancer Res 11(2):R16。
  4. Listenberger,L.L。和Brown,D.A。(2007)。 荧光检测脂滴和相关蛋白。 Curr Protoc Cell Biol Chapter 24:Unit 24 22.
  5. Liu,H.,Liu,J.Y.,Wu,X。和Zhang,J.T。(2010)。 脂肪酸合酶的生物化学,分子生物学和药理学,新兴治疗靶标和诊断/预后标记。 Int J Biochem Mol Biol 1(1):69-89
  6. Ranall,M.V.,Gabrielli,B.G.and Gonda,T.J。(2011)。 含1,6-二苯基己三烯的中性脂滴的高含量成像。 Biotechniques 51(1):35-36,38-42。
  7. Walther,T.C.和Farese,R.V.,Jr。(2009)。 脂滴的生命。 Biochim Biophys Acta 1791 (6):459-466。
  8. Wang,X.,Sun,Y.,Wong,J.和Conklin,D.S.(2013)。 PPARgamma维持ERBB2阳性乳腺癌干细胞 癌基因 32(49):5512-552。
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引用:Baumann, J. M., Kokabee, L., Wang, X., Sun, Y., Wong, J. and Conklin, D. S. (2015). Metabolic Assays for Detection of Neutral Fat Stores. Bio-protocol 5(12): e1511. DOI: 10.21769/BioProtoc.1511.
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Alberto Daniel
Instituto de Investigaciones Biomédicas, UNAM
Hi, I'm trying to use sodium palmitate in culture and the question is if in your treatment protocol did you used sodium palmitate conjugated whit albumin, or it's feasible to use palmitate dissolved in fetal bovine serum and medium.
Thank you for your cooperation
Best regards
2/17/2017 2:43:11 PM Reply