FACS-based Glucose Uptake Assay of Mouse Embryonic Fibroblasts and Breast Cancer Cells Using 2-NBDG Probe

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The Journal of Biological Chemistry
Oct 2017



This is a flow cytometry-based protocol to measure glucose uptake of mouse embryonic fibroblasts (MEFs) and breast cancer cells in vitro. The method is a slightly modified and updated version as previously described (Dong et al., 2017). Briefly, the target cells are incubated with the fluorescently tagged 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) for 2 h or 30 min, and the efficiency of glucose uptake is examined using a flow cytometer. This method can be adapted to measure a variety of adipocytes, immune cells, MEFs and cancer cells.

Keywords: Mouse embryonic fibroblasts (MEFs) (小鼠胚胎成纤维细胞(MEFs)), Glucose uptake (葡萄糖摄取), 2-NBDG (2-NBDG), Cancer cells (癌症细胞), Flow cytometry (流式细胞术)


Glucose is the primary source of energy for cells. A family of glucose transporters (GLUT) is responsible for transporting glucose across cell membranes (Kohn et al., 1996). Changes in glucose uptake can reflect the changes in cellular metabolism. For example, tumor cells generally use glucose for aerobic glycolysis in order to support their rapid proliferation. Normally, tumor cells have increased rates of glucose uptake compared to normal cells (Vander Heiden et al., 2009). The 2-deoxyglucose (2DG) is a glucose analog and it accumulates in the cell as 2-deoxyglucose-6-phosphate (2DG6P). 2DG6P has been a gold standard for measuring glucose uptake for a long time (Yamamoto et al., 2011). Although the measurement of radio-labeled 2DG6P is sensitive, many researchers avoid this method because the handling and disposal of radioactive material require a special procedure.

Another non-metabolizable glucose analog is the fluorescently tagged 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG). This molecule accumulates in living cells through a glucose transporter and does not enter the glycolytic pathway. Fluorescence generated by 2-NBDG is proportional to glucose uptake. 2-NBDG fluorescence typically displays excitation/emission maxima of ~465/540 nm. It can be detected using optical filters designed for fluorescein using flow cytometry (O'Neil et al., 2005; Zou et al., 2005; Nitin et al., 2009).

Materials and Reagents

  1. Pipette tips 200 μl tips (USA Scientific, catalog number: 1111-1800 )
  2. 10 cm Petri dishes (Corning, catalog number: 430167 )
  3. 15 ml polystyrene centrifuge tubes (Corning, Falcon®, catalog number: 352097 )
  4. 5 ml round-bottom polystyrene tubes with cell-strainer cap (Corning, Falcon®, catalog number: 352235 ), 0.35 µm nylon mesh
  5. Embryos of C57BL/6 WT mouse (13.5 days)
  6. MCF7 breast cancer cells (ATCC, catalog number: HTB-22 )
  7. Mouse embryonic fibroblasts (MEFs)
  8. 0.05% trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25300062 )
  9. 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) (Thermo Fisher Scientific, InvitrogenTM, catalog number: N13195 )
  10. Dulbecco’s modified Eagle medium (DMEM), high glucose (GE Healthcare, Hyclone, catalog number: SH30243.01 )
  11. Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 100-106 )
  12. Penicillin/streptomycin (Pen/Strep) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  13. 2-Mercaptoethanol (2-Me) (Sigma-Aldrich, catalog number: M6250-500ML )
  14. Sodium chloride (NaCl)
  15. Potassium chloride (KCl)
  16. Sodium phosphate dibasic (Na2HPO4)
  17. Potassium phosphate monobasic (KH2PO4)
  18. DMEM culture medium (see Recipes)
  19. Phosphate-buffered saline (PBS, 1x) (see Recipes)
  20. FACS buffer (see Recipes)


  1. Pipettes (Gilson, model: P200, catalog number: F123601 )
  2. Refrigerated centrifuge (Eppendorf, model: 5810 R )
  3. Hemocytometer chamber (Hausser Scientific)
  4. Water bath (Fisher Scientific, model: IsotempTM 205 )
  5. Inverted microscope (Nikon Instruments, model: Eclipse Ts2-FL )
  6. Autoclave
  7. Cell culture hood (Thermo Fisher Scientific, Forma class II, A2)
  8. Cell culture incubator (Thermo Fisher Scientific, Forma series II)
  9. FACSCalibur flow cytometer (BD Biosciences)


  1. FlowJo software version 10.0.8 or newer (FlowJo)


  1. 2-NBDG uptake assay for MEFs
    1. Mouse embryonic fibroblasts (MEFs) are isolated from the embryos of C57BL/6 WT mouse (13.5 days) (Dong et al., 2015).
    2. Culture the MEF cells until reaching 80-90% confluence in 10 cm Petri dishes with DMEM growth medium in a humidified cell culture incubator (37 °C, 5% CO2).
      Note: Don’t use MEFs beyond passage 3. MEFs usually become senescent at about passage 4 to 5.
    3. Remove culture medium and wash cells one time with 10 ml 1x PBS.
    Note: Use room temperature or pre-warmed 1x PBS from Step A3 to Step A9. Using chilled 1x PBS after Step A9.
    1. Trypsinize cells using 4 ml of 0.05% trypsin-EDTA for 3 min at 37 °C.
    2. Transfer cells to 15 ml polystyrene centrifuge tubes.
    3. Harvest cells at 200 x g for 5 min by centrifugation.
    4. Wash pelleted cells one time with 5 ml 1x PBS.
    5. Count cells using a hemocytometer chamber.
    6. Incubate 1 x 106 MEF cells in a 37 °C water bath for 2 h with 1 ml of PBS containing 100 μM 2-NBDG. Incubate the same number of MEFs in the water bath with 1 ml PBS without 2-NBDG as a negative control.
    7. Pellet the cells at 200 x g for 5 min by centrifugation. After washing the cells with chilled 1x PBS, the cells are pelleted at 200 x g for 5 min by centrifugation.
    8. Resuspend cells in 0.5 ml of ice-cold 1x PBS with 2% FBS.
      Note: Always keep cells on ice after this step.
    9. Filter cells through a 35 µm nylon mesh (the cell-strainer cap of the 5 ml round-bottom polystyrene tubes) to obtain a uniform single-cell suspension in a 5 ml tube.
    10. Keep the samples on ice until analysis on a flow cytometer.
    11. Perform flow cytometric analysis on a FACSCalibur flow cytometer. Acquire 10,000 single-cell events per reaction.
    12. Analyze fluorescence intensity using FlowJo software.

  2. 2-NBDG uptake assay for breast cancer cells
    Using the same procedure as MEFs’ uptake assay except incubating 1 x 106 MCF7 cells in a 37 °C water bath only for 30 min (instead of two hours for MEFs) with 1 ml of PBS containing 100 μM 2-NBDG.
    Note: Cancer cells increase glycolysis even in the presence of adequate oxygen. To compensate the reduced energy yield due to aerobic glycolysis, there is massive glucose uptake in cancer cells. Glucose transporters (GLUTs) are responsible for constitutive transportation of glucose into cells. Breast tumor and the other tumor cell lines showed higher expression levels of GLUTs 1 to 4 compared to mouse fibroblasts (Maddalena et al., 2015). This is one possible reason why cancer cells have the higher capacity of retaining glucose compared to normal fibroblasts. The other reasons include mitochondria defects and altered PI3K–Akt–mTOR signaling pathway in cancer cells.

Data analysis

True uptake of 2-NBDG-labeled cells is determined using a gating strategy that allows analysis of live cells. 10,000 single cells were collected. We set a gate (R1) on the FSC-SSC plot to select the interested cell population but exclude cell debris. We then displayed the relative fluorescence of the gated cells on the x-axis and the number of cell count on the y-axis. The 2-NBDG positive cells are analyzed by plotting histograms vs. FITC as Figure 1.

Figure 1. Glucose uptake by MEFs and MCF7 breast cancer cells. A. WT MEFs were assessed for glucose uptake by incorporation of a fluorescent glucose analog 2-NBDG for 2 h. B. MCF7 cells were assessed for glucose uptake by 2-NBDG for 30 min. The fluorescence was detected in the FL-1 (green fluorescence) channel using FACSCalibur and the results are shown as histograms. For quantitation and statistical analysis, we normally calculate the mean value of FL2 fluorescence intensity from triplicate experiments.


To prevent 2-NBDG leaking from cell membrane, samples should always be kept on ice after stopping the 2-NBDG uptake reaction. Do not keep the samples on ice for more than 2 h before analyzing on the flow cytometer.


  1. DMEM culture medium
    10% heat-inactivated FBS
    1% of penicillin/streptomycin
    50 µM 2-mercaptoethanol (2-Me)
    Note: Store at 4 °C; warm to 37 °C before use.
  2. Phosphate-buffered saline (PBS, 1x)

    Adjust the pH to 7.4 and then fill distilled water to a final volume of 1 L. Autoclave for 15 min and store at room temperature
  3. FACS buffer
    1 x PBS
    2% FBS
    Note: Prepare just before use and keep on ice.


We thank Constance Porretta, who is technical director of Flow Cytometry Shared Resource in Louisiana State University Health Science Center (LSUHSC), for the help with FACS experiments. The authors declare no conflicts of interest. This work was supported by funds from LSUSHC School of Medicine and Fred G, Brazda Foundation. This is an elaborated method based on the work published by Dong et al. (2017).


  1. Dong, S., Baranwal, S., Garcia, A., Serrano-Gomez, S. J., Eastlack, S., Iwakuma, T., Mercante, D., Mauvais-Jarvis, F. and Alahari, S. K. (2017). Nischarin inhibition alters energy metabolism by activating AMP-activated protein kinase. J Biol Chem 292(41): 16833-16846.
  2. Dong, S., Maziveyi, M. and Alahari, S. K. (2015). Primary tumor and MEF cell isolation to study lung metastasis. J Vis Exp (99): e52609.
  3. Kohn, A. D., Summers, S. A., Birnbaum, M. J. and Roth, R. A. (1996). Expression of a constitutively active Akt Ser/Thr kinase in 3T3-L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation. J Biol Chem 271(49): 31372-31378.
  4. Maddalena, F., Lettini, G., Gallicchio, R., Sisinni, L., Simeon, V., Nardelli, A., Venetucci, A. A., Storto, G. and Landriscina, M. (2015). Evaluation of glucose uptake in normal and cancer cell lines by positron emission tomography. Mol Imaging 14: 490-8.
  5. Nitin, N., Carlson, A. L., Muldoon, T., El-Naggar, A. K., Gillenwater, A. and Richards-Kortum, R. (2009). Molecular imaging of glucose uptake in oral neoplasia following topical application of fluorescently labeled deoxy-glucose. Int J Cancer 124(11): 2634-2642.
  6. O'Neil, R. G., Wu, L. and Mullani, N. (2005). Uptake of a fluorescent deoxyglucose analog (2-NBDG) in tumor cells. Mol Imaging Biol 7(6): 388-392.
  7. Vander Heiden, M. G., Cantley, L. C. and Thompson, C. B. (2009). Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324(5930): 1029-1033.
  8. Yamamoto, N., Ueda, M., Sato, T., Kawasaki, K., Sawada, K., Kawabata, K. and Ashida, H. (2011). UNIT 12.14 Measurement of glucose uptake in cultured cells. In: Enna, S. J. (Ed.) Curr Protoc Pharmacol, Chapter 12, 11-22.
  9. Zou, C., Wang, Y. and Shen, Z. (2005). 2-NBDG as a fluorescent indicator for direct glucose uptake measurement. J Biochem Biophys Methods 64(3): 207-215.


这是一种基于流式细胞术的方法,用于在体外测量小鼠胚胎成纤维细胞(MEFs)和乳腺癌细胞的葡萄糖摄取。 该方法是稍微修改和更新的版本(Dong等人,2017)。 简言之,将靶细胞与荧光标记的2-(N-(7-硝基苯-2-基-1,3-二唑-4-基)氨基)-2-脱氧葡萄糖(2-NBDG)一起温育2小时或 30分钟,并使用流式细胞仪检查葡萄糖摄取的效率。 该方法可适用于测量各种脂肪细胞,免疫细胞,MEF和癌细胞。

【背景】葡萄糖是细胞的主要能量来源。葡萄糖转运蛋白家族(GLUT)负责跨葡萄糖转运葡萄糖(Kohn等人,1996)。葡萄糖摄取的变化可以反映细胞代谢的变化。例如,肿瘤细胞通常使用葡萄糖进行有氧糖酵解以支持其快速增殖。通常,与正常细胞相比,肿瘤细胞具有增加的葡萄糖摄取速率(Vander Heiden等人,2009)。 2-脱氧葡萄糖(2DG)是一种葡萄糖类似物,它以2-脱氧葡萄糖-6-磷酸(2DG6P)的形式积累在细胞中。 2DG6P长期以来一直是测量葡萄糖摄取的黄金标准(Yamamoto et al。,2011)。尽管放射性标记的2DG6P的测量是敏感的,但许多研究人员避免了这种方法,因为放射性物质的处理和处置需要特殊的程序。

另一种不可代谢的葡萄糖类似物是荧光标记的2-(N-(7-硝基苯-2-氧杂-1,3-二唑-4-基)氨基)-2-脱氧葡萄糖(2-NBDG)。该分子通过葡萄糖转运蛋白积聚在活细胞中,不会进入糖酵解途径。 2-NBDG产生的荧光与葡萄糖摄取成正比。 2-NBDG荧光通常显示〜465 / 540nm的激发/发射最大值。它可以使用流式细胞术使用设计用于荧光素的光学滤光片来检测(O'Neil等人,2005; Zou等人,2005; Nitin等人。,2009)。

关键字:小鼠胚胎成纤维细胞(MEFs), 葡萄糖摄取, 2-NBDG, 癌症细胞, 流式细胞术


  1. 移液器吸头200μl吸头(USA Scientific,目录号:1111-1800)
  2. 10厘米培养皿(康宁,目录号:430167)
  3. 15毫升聚苯乙烯离心管(Corning,Falcon,目录号:352097)
  4. 5毫升圆底聚苯乙烯管与细胞过滤器盖(康宁,猎鹰®,目录号:352235),0.35微米尼龙网格

  5. C57BL / 6 WT小鼠胚胎(13.5天)
  6. MCF7乳腺癌细胞(ATCC,目录号:HTB-22)
  7. 小鼠胚胎成纤维细胞(MEFs)
  8. 0.05%胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM,目录号:25300062)
  9. 2-(N-(7-硝基苯-2-氧杂-1,3-二唑-4-基)氨基)-2-脱氧葡萄糖(2-NBDG)(Thermo Fisher Scientific,Invitrogen TM,目录号:N13195)
  10. Dulbecco改良Eagle培养基(DMEM),高葡萄糖(GE Healthcare,Hyclone,目录号:SH30243.01)
  11. 胎牛血清(FBS)(Gemini生物产品,目录号:100-106)
  12. 青霉素/链霉素(Pen / Strep)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  13. 2-巯基乙醇(2-Me)(Sigma-Aldrich,目录号:M6250-500ML)
  14. 氯化钠(NaCl)
  15. 氯化钾(KCl)
  16. 磷酸氢二钠(Na 2 HPO 4)
  17. 磷酸二氢钾(KH 2 PO 4)
  18. DMEM培养基(见食谱)
  19. 磷酸盐缓冲盐水(PBS,1x)(见食谱)
  20. FACS缓冲液(见食谱)


  1. 移液器(Gilson,型号:P200,目录号:F123601)
  2. 冷冻离心机(Eppendorf,型号:5810 R)
  3. 血细胞计数器室(Hausser科学)
  4. 水浴(Fisher Scientific,型号:Isotemp TM 205)
  5. 倒置显微镜(尼康仪器,型号:Eclipse Ts2-FL)
  6. 高压灭菌器
  7. 细胞培养罩(Thermo Fisher Scientific,Forma class II,A2)
  8. 细胞培养孵化器(Thermo Fisher Scientific,Forma系列II)
  9. FACSCalibur流式细胞仪(BD Biosciences)


  1. FlowJo软件版本10.0.8或更高版本(FlowJo)


  1. 用于MEF的2-NBDG摄取测定
    1. 从C57BL / 6WT小鼠的胚胎中分离小鼠胚胎成纤维细胞(MEF)(13.5天)(Dong等人,2015)。
    2. 培养MEF细胞直至在加湿细胞培养箱(37℃,5%CO 2)中用DMEM生长培养基在10cm培养皿中达到80-90%汇合。
    3. 取出培养基并用10ml 1x PBS洗涤细胞一次。
      注意:使用室温或从步骤A3到步骤A9的预热1x PBS。步骤A9后使用冷冻的1x PBS。

    4. 使用4毫升0.05%胰蛋白酶-EDTA胰蛋白酶消化细胞3分钟
    5. 将细胞转移到15毫升聚苯乙烯离心管中。
    6. 通过离心将200gxg的细胞收获5分钟。

    7. 一次用5 ml 1x PBS清洗沉淀的细胞
    8. 使用血细胞计数器计数细胞。
    9. 在37℃水浴中用1ml含有100μM2-NBDG的PBS孵育1×10 6个MEF细胞2小时。孵育相同数量的MEFs在水浴中使用1 ml PBS而不使用2-NBDG作为阴性对照。
    10. 通过离心将细胞沉淀200g×g 5分钟。用冷冻的1x PBS洗涤细胞后,通过离心将细胞沉淀200g×5分钟。
    11. 将细胞重悬于0.5 ml冰冷的含2%FBS的1x PBS中。
    12. 过滤细胞通过35微米尼龙网(<5毫升圆底聚苯乙烯管的细胞过滤器帽),以获得均匀的单细胞悬浮液在5毫升管中。
    13. 保持样品在冰上,直到在流式细胞仪上分析。
    14. 在FACSCalibur流式细胞仪上进行流式细胞分析。每次反应可获得10,000个单细胞事件。
    15. 使用FlowJo软件分析荧光强度。

  2. 乳腺癌细胞的2-NBDG摄取检测
    使用与MEFs摄取测定相同的程序,除了在37℃水浴中将1×10 6 MCF7细胞与1ml含有100μlPBS的PBS一起孵育30分钟(而不是2小时用于MEFs) μM2-NBDG。



图1. MEF和MCF7乳腺癌细胞对葡萄糖的摄取A.通过掺入荧光葡萄糖类似物2-NBDG 2小时评估WT WT MEF的葡萄糖摄取。 B.通过2-NBDG评估MCF7细胞葡萄糖摄取30分钟。使用FACSCalibur在FL-1(绿色荧光)通道中检测荧光,结果显示为直方图。对于定量和统计分析,我们通常计算一式三份实验中FL2荧光强度的平均值。




  1. DMEM培养基
  2. 磷酸盐缓冲盐水(PBS,1x)

  3. FACS缓冲液
    1 x PBS


我们感谢路易斯安那州立大学健康科学中心(LSUHSC)Flow Cytometry Shared Resource技术总监Constance Porretta帮助进行FACS实验。作者宣称没有利益冲突。这项工作得到了LSUSHC医学院和Fred G,Brazda基金会的资助。这是一个基于Dong等人发表的工作的详细方法。(2017)。


  1. Dong,S.,Baranwal,S.,Garcia,A.,Serrano-Gomez,S.J.,Eastlack,S.,Iwakuma,T.,Mercante,D.,Mauvais-Jarvis,F.和Alahari,S.K。(2017)。 Nischarin抑制剂通过激活AMP激活的蛋白激酶改变能量代谢。 J Biol Chem 292(41):16833-16846。
  2. Dong,S.,Maziveyi,M.and Alahari,S.K。(2015)。 原发性肿瘤和MEF细胞分离以研究肺部转移。 J Vis Exp (99):e52609。
  3. Kohn,A. D.,Summers,S.A.,Birnbaum,M.J。和Roth,R.A。(1996)。 3T3-L1脂肪细胞中组成型活化的Akt Ser / Thr激酶的表达刺激葡萄糖摄取和葡萄糖转运蛋白4易位。 J Biol Chem 271(49):31372-31378。
  4. Maddalena,F.,Lettini,G.,Gallicchio,R.,Sisinni,L.,Simeon,V.,Nardelli,A.,Venetucci,A. A.,Storto,G.和Landriscina,M.(2015年)。 通过正电子发射断层扫描评估正常和癌细胞系的葡萄糖摄取 Mol Imaging 14:490-8。
  5. Nitin,N.,Carlson,A.L.,Muldoon,T.,El-Naggar,A.K。,Gillenwater,A。和Richards-Kortum,R。(2009)。 在局部应用荧光标记的脱氧葡萄糖后口腔肿瘤中葡萄糖摄取的分子成像 Int J Cancer 124(11):2634-2642。
  6. O'Neil,R. G.,Wu,L.和Mullani,N。(2005)。 摄取肿瘤细胞中的荧光脱氧葡萄糖类似物(2-NBDG) Mol Imaging Biol 7(6):388-392。
  7. Vander Heiden,M. G.,Cantley,L. C.和Thompson,C. B.(2009)。 了解Warburg效应:细胞增殖的代谢需求 科学 324(5930):1029-1033。
  8. Yamamoto,N.,Ueda,M.,Sato,T.,Kawasaki,K.,Sawada,K.,Kawabata,K。和Ashida,H。(2011)。 单元12.14培养细胞中葡萄糖摄取的测量 Enna,SJ(Ed。) Curr Protoc Pharmacol ,第12,11-22。
  9. Zou,C.,Wang,Y.和Shen,Z。(2005)。 2-NBDG作为直接葡萄糖摄取测量的荧光指示剂。 J Biochem Biophys Methods 64(3):207-215。
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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. Dong, S. and Alahari, S. (2018). FACS-based Glucose Uptake Assay of Mouse Embryonic Fibroblasts and Breast Cancer Cells Using 2-NBDG Probe. Bio-protocol 8(8): e2816. DOI: 10.21769/BioProtoc.2816.
  2. Dong, S., Baranwal, S., Garcia, A., Serrano-Gomez, S. J., Eastlack, S., Iwakuma, T., Mercante, D., Mauvais-Jarvis, F. and Alahari, S. K. (2017). Nischarin inhibition alters energy metabolism by activating AMP-activated protein kinase. J Biol Chem 292(41): 16833-16846.