Measurement of Glucose Uptake in Cultured Cells

Norio Yamamoto1, Manabu Ueda‐Wakagi2, Takuya Sato1, Kengo Kawasaki1, Keisuke Sawada3, Kyuichi Kawabata4, Mitsugu Akagawa5, Hitoshi Ashida3

1 Research & Development Institute, House Wellness Foods Corporation, Hyogo, 2 National Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, 3 Department of Agrobiosciences, Graduate School of Agricultural Science, Kobe University, Kobe, 4 Department of Bioscience, Fukui Prefectural University, Fukui, 5 Department of Biological Chemistry, Division of Applied Life Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 12.14
DOI:  10.1002/0471141755.ph1214s71
Online Posting Date:  December, 2015
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Abstract

Facilitative glucose uptake transport systems are ubiquitous in animal cells and are responsible for transporting glucose across cell surface membranes. Evaluation of glucose uptake is crucial in the study of numerous diseases and metabolic disorders such as myocardial ischemia, diabetes mellitus, and cancer. Detailed in this unit are laboratory methods for assessing glucose uptake into mammalian cells. The unit is divided into five sections: (1) a brief overview of glucose uptake assays in cultured cells; (2) a method for measuring glucose uptake using radiolabeled 3‐O‐methylglucose; (3) a method for measuring glucose uptake using radiolabeled 2‐deoxyglucose (2DG); (4) a microplate method for measuring 2DG‐uptake using an enzymatic, fluorometric assay; and (5) a microplate‐based method using a fluorescent analog of 2DG. © 2015 by John Wiley & Sons, Inc.

Keywords: glucose uptake; cell‐based assay; 3‐O‐methylglucose; 2‐deoxyglucose; radioisotope; nonradioisotope; 2‐NBDG,fluorescence

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Glucose Uptake Using Radiolabeled 3‐O‐Methylglucose
  • Basic Protocol 2: Measurement of Glucose Uptake Using Radiolabeled 2‐Deoxyglucose
  • Basic Protocol 3: Measurement of Nonradiolabeled 2‐Deoxyglucose Uptake
  • Basic Protocol 4: Measurement of Glucose Uptake Using a Fluorescent Derivative of 2‐Deoxyglucose, 2‐NBDG
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Glucose Uptake Using Radiolabeled 3‐O‐Methylglucose

  Materials
  • Cells [e.g., 3T3‐L1 mouse adipose cells (ATCC #CL‐173), L6 rat skeletal muscle cells (ATCC #CRL‐1458), or primary adipose or muscle cells isolated from live animals]
  • Appropriate culture medium
  • Test sample(s), such as synthetic compound(s), natural products and positive controls, such as insulin
  • KRH buffer containing 0.1% BSA (see recipe)
  • Dimethyl sulfoxide (DMSO)
  • Cytochalasin B stock solution (see recipe)
  • Stock solution of radioactive 3MG (see recipe)
  • 0.05 M NaOH
  • Commercial scintillation fluid (e.g., Clear‐sol I, xylene‐based liquid scintillation cocktail, Nacalai Tesque, cat. no. 09135‐93)
  • Cell culture plate, 24‐well format (e.g., Corning, cat. no. 3338; Costar, cat. no. 3548)
  • Plate mixer (e.g., IKA, MS‐3 digital)
  • Polyethylene scintillation vials (e.g., Fisher Scientific, cat. no 03‐337‐34)
  • Liquid scintillation counter (e.g., Aloca, LSC‐600B)
  • Vortex mixer (e.g., Scientific Industries, Model No. SI‐0286)
  • Additional reagents and equipment for protein assay ( appendix 3A; Olson and Markwell, )
NOTE: All solutions and equipment that come into contact with cells must be sterile, and proper aseptic technique used when working with cell cultures.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: Measurement of Glucose Uptake Using Radiolabeled 2‐Deoxyglucose

  Materials
  • Cells [e.g., 3T3‐L1 mouse adipose cells (ATCC #CL‐173), L6 rat skeletal muscle cells (ATCC #CRL‐1458), or primary adipose or muscle cells isolated from live animals]
  • Appropriate culture medium
  • KRH buffer containing 0.1% BSA (see recipe)
  • Test sample(s), such as synthetic compound(s), natural products and positive controls, such as insulin
  • Dimethyl sulfoxide (DMSO)
  • Cytochalasin B stock solution (see recipe)
  • Radioactive 2DG stock solution (see recipe)
  • 0.05 M NaOH
  • Commercial scintillation fluid (e.g., Clear‐sol I, xylene‐based liquid scintillation cocktail, Nacalai Tesque, cat. no. 09135‐93)
  • Cell culture plate, 24‐well format (e.g., Corning, cat. no. 3338; Costar, cat. no. 3548)
  • Plate mixer (e.g., IKA, MS‐3 digital)
  • Polyethylene scintillation vials (e.g., Fisher Scientific, cat. no 03‐337‐34)
  • Liquid scintillation counter (e.g., Aloca, cat. no. LSC‐600B)
  • Vortex mixer (Scientific Industries, model no. SI‐0286)
NOTE: All solutions and equipment that come into contact with cells must be sterile, and proper aseptic technique used for work with cell cultures.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 3: Measurement of Nonradiolabeled 2‐Deoxyglucose Uptake

  Materials
  • Cells [e.g., 3T3‐L1 mouse adipose cells (ATCC #CL‐173), L6 rat skeletal muscle cells (ATCC #CRL‐1458), and primary adipose or muscle cells isolated from live animals
  • Appropriate culture medium (containing FBS)
  • Serum‐free culture medium
  • Dimethyl sulfoxide (DMSO)
  • KRH buffer containing 0.1% BSA (see recipe)
  • Test sample(s), such as synthetic compound(s), natural products and positive controls, such as insulin
  • 2‐Deoxy‐D‐glucose (2DG; Sigma‐Aldrich, cat. no. D6134)
  • 0.1 M NaOH
  • 0.1 M HCl
  • 200 mM and 50 mM TEA buffer, pH 8.1 (see recipe)
  • Assay solution (see recipe)
  • 2‐Deoxy‐D‐glucose 6‐phosphate sodium salt, monohydrate (DG6P; Santa Cruz, cat. no. SC‐220734; Carbosynth, cat. no. MD06697)
  • Cell culture plate: 96‐well format (e.g., Corning, cat. no. 3997) or 24‐well format (e.g., Corning, cat. no. 3338)
  • Microplate mixer (e.g., IKA, MS‐3 digital)
  • Constant temperature water bath at 60° to 85°C (dry block heat baths with microtiter blocks or laboratory heating ovens are also acceptable)
  • Black or white assay plate: 96‐well format (e.g., Corning, cat. no. 3991 or cat. no. 3912) or 384‐well format (Corning, cat. no. 3573 or cat. no. 3572)
  • Fluorescence microplate reader to measure fluorescence (λ ex = 530 to 570 nm, λ em = 590 to 620 nm; optimal filters or wavelengths are λ ex = 530 to 560 nm, λ em = 590 nm)
NOTE: All solutions and equipment that come into contact with cells must be sterile, and proper aseptic technique used for work with cell cultures.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 4: Measurement of Glucose Uptake Using a Fluorescent Derivative of 2‐Deoxyglucose, 2‐NBDG

  Materials
  • Cells [e.g., L6 rat skeletal muscle cells (ATCC #CRL‐1458), C2C12 mouse myoblast cells (ATCC #CRL‐1772), COS‐7 African green monkey kidney cells (ATCC #CRL‐1651), or primary adipose or muscle cells isolated from live animals]
  • Appropriate culture medium
  • Test sample(s), such as synthetic compound(s) or natural products, and positive controls, such as insulin
  • Serum‐free medium, containing 1.0 g/liter glucose (e.g,. Nakalai Tesque, cat. no. 08490‐05)
  • Dimethylsulfoxide (DMSO)
  • Glucose‐free medium, without FBS (e.g., Nakalai Tesque, cat. no. 09891‐25)
  • 2‐NBDG stock solution (see recipe)
  • Cytochalasin B stock solution (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Potassium phosphate buffer, pH 10 ( appendix 2A)
  • Triton X‐100
  • Cell culture plate, 96‐well black, clear‐bottom format (e.g., Corning, cat. no. 356700)
  • Plate mixer (e.g., IKA, MS‐3 digital)
  • Fluorescence microplate reader to measure fluorescence (λ ex = 460 to 490 nm, λ em = 530 to 550 nm; optimal wavelengths are λ ex = 467 nm, λ em = 542 nm); the same measurement parameters as for fluorescein measurement can be applied
NOTE: All solutions and equipment that come into contact with cells must be sterile, and proper aseptic techniques must be used for work with cell cultures.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.
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Figures

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Literature Cited

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Key References
  Leira et al., 2002. See above.
  This article is the first report of a fluorometric microplate assay to assess glucose uptake by using a fluorescent derivative, 2‐NBDG.
  Mueckler and Thorens, 2013. See above.
  This review summarizes the knowledge of the identified members of the glucose transporter family, proposes a basis for their diversity highlighting differences in kinetic properties, substrate specificity, and hormonal regulation, and discusses aberrant expression and/or dysfunction of these transporters in disease states.
  Sokoloff et al., 1977. See above.
  A radioautographic method to measure regional rates of brain glucose consumption with 2‐[14C]deoxyglucose.
  Yamamoto et al., 2006. See above.
  A detailed description concerning mechanism of a nonradioisotopic 2DG‐uptake assay.
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