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2-nbdg Glucose Uptake Assay

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Preparation Of Working Solution

Add 5 µL of 2-NBDG to 1.5 mL of Assay Buffer I and mix well to make 2-NBDG staining solution. Protect from light. Note: This 2-NBDG staining solution is stable for 1 hour at room temperature. As the optimal staining conditions may vary depending on different cell types, its recommended to determine the optimal concentration of Component A for each specific experiment.

For guidelines on cell sample preparation, please visit

Effects Of Starving Hk

Prior to the glucose uptake measurement, moderate starvation without glucose supplements results in an increased glucose uptake into cells, in general. To optimize the best readout for glucose uptake of the HK-2 cells, we next incubated the cells in glucose-free DMEM for 0, 1 h or 5 h before adding the buffer supplemented with 2-NBDG, and then followed by the treatment with regular buffer for our glucose uptake assay as in Fig. 4. Surprisingly, both total glucose uptake and Na+-independent ) glucose uptake are relatively stable among 0, 1 h and 5 h starvations of the cells, suggesting no starvation is required for the SGLT2-mediated glucose uptake assay in HK-2 cells.

Fig. 4

Effects of Starving HK-2 cells on glucose uptake. HK-2 cells were incubated in DMEM without glucose for 0, 1 and 5 h, then 2-NBDG uptake assay was performed in Na+ or Na+ buffer. The uptake values were normalized to the fluorescence intensity of Na+ group without starvation. Experiments were performed in triplicate and data are expressed as mean±S.E.M of three independent experiments

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Optimization Of Incubation Time And Concentration Of 2

For the best fluorescence of 2-NBDG detected by microscope, HK-2 cells were incubated with 200 M 2-NBDG in Na+ or Na+-free buffer for 5, 10, 15, 30, 60, 90, 120 or 150 min respectively, followed by measuring the fluorescence intensity of 2-NBDG in the cells. The quantification in Fig. 3a reflected the increasing glucose uptake in HK-2 cells in the processing of the more incubation time. Particularly, the glucose uptake reached the maximum by the 60 min of the 2-NBDG incubation.

Fig. 3

Optimization of incubation time and concentration of 2-NBDG. a HK-2 cells were incubated with 200 M 2-NBDG for 5, 10, 15, 30, 60, 90, 120 or 150 min in Na+ or Na+ buffer. SGLT2-mediated 2-NBDG uptake was calculated by subtracting the intensity of the Na+ to Na+ group. b HK-2 cells were incubated with 10, 50, 100, 200, 400, 600, 800, 1000 or 1200 M 2-NBDG for 60 min in Na+ or Na+ buffer, and then SGLT2-mediated 2-NBDG uptake was calculated as above. Experiments were performed in triplicate and data are represented as mean±S.E.M. of three independent experiments

Traditional Glucose Uptake Methods

Frontiers

Available glucose uptake assays differ by the substrate for transport. Since glucose itself is shuttled into a variety of pathways after transport, non-metabolizable glucose analogs must be used instead. One type is the fluorescently tagged 2-NBDG amino)-2-deoxyglucose). This probe accumulates in the cell and works well for imaging, but because it is much larger than glucose, there is concern that it is not transported in the same way and hence may not be an accurate indicator of glucose transporter activity. Another glucose analog is 2DG . This molecule gets transported and phosphorylated just like glucose, but since it is not acted upon significantly by other cellular enzymes, it accumulates in the cell as 2DG6P .

Assay
  • Requires handling and disposal of radioactive materials

Fluorescence

  • Requires multiple sample preparation steps
  • Narrow window of detection
  • Can detect very low 2DG6P
  • Requires multiple sample preparation steps
  • Narrow window of detection

Intracellular accumulation of a fluorescently labelled glucose analog

  • Works well for imaging
  • Doesn’t work well in plate formats
  • May not correctly reflect transporter activity

Luminescence

  • Can detect very low 2DG6P with large assay window
  • Direct in-well detection with no wash steps
  • Scalable for high throughput
  • Not applicable for cell imaging

Figure 1. Comparison of enzymatic detection assays.Figure 2. Comparison of radioactive and luminescent assays.

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Efficient And Modified 2

J Pharmacol Toxicol MethodsMay-Jun 2021 109:107069.PMID: 33892108DOI: 10.1016/j.vascn.2021.107069

Under type-2 diabetes, insulin resistance develops in skeletal muscles as a key defect and to study the disorder, its manifestation, and possible solution, measurement of glucose uptake is a fundamental necessity. Of various approaches fluorescent labelled glucose analogue, 2-NBDG solution is the most popular one. Although 2-NBDG based assay is the most widely used approach in various cells including skeletal muscle, even then all available protocols possess huge variability which impacts the overall data reproducibility. Moreover, starvation , one of the prerequisite condition for glucose uptake assay, itself induces stress specifically during longer pre-incubation periods and alters muscle cell metabolism and morphology, but the fact has not been duly considered. Therefore in the present article, using specific skeletal muscle cells i.e. C2C12 myotubes, we have re-established the conditions like pre-incubation time period, concentrations of insulin, glucose and serum/BSA while maintaining the cultured myotubes in morphologically healthy state. Our lab standardized protocols were observed to be effective in studying insulin resistance condition induced by diverse stresses in myotubes. Comparative study conducted with already established protocols demonstrates that the present method is more efficient, effective and better improvised for studying glucose uptake in C2C12.

Sglt2 Inhibitors Dapagliflozin And Phlorizin Block 2

To address the inhibition effects of SGLT2 inhibitors on 2-NBDG uptake in our screening assay is reliable and similar to traditional methods, we treated HK-2 cells with two SGLT2 inhibitors Dapagliflozin and Phlorizin and then performed the 2-NBDG uptake assay. As we predicted, Dapagliflozin strongly inhibited Na+-dependent glucose uptake by reducing 43.7% of 2-NBDG uptake, whereas Phlorizin induced 30.2% decrease in Na+-dependent glucose uptake . These positive results constitute a good starting point for assay development.

Fig. 5

SGLT2 inhibitors Dapagliflozin and Phlorizin blocks 2-NBDG uptake in HK-2 cells. a Inhibitory effect of Dapagliflozin or Phlorizin on 2-NBDG uptake in HK-2 cells. Values were normalized to the fluorescence intensity of 2-NBDG uptake dissolved in Na+ buffer. Experiments were performed in triplicate and data are presented as mean±S.E.M. of three independent experiments. *P< 0.05 versus Na+-DMSO #P< 0.05 versus Na+-ethanol. b Inhibition ratio of Dapagliflozin and Phlorizin derived from . c 2-NBDG uptake was measured in HK-2 cells incubated with Dapagliflozin for 0.167, 0.5, 1, 2, 3, 4 and 5 h. d Dose-dependent response curve of Dapagliflozin on 2-NBDG uptake assay of HK-2 cells

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Endogenous Sglt2 Of Hk

To study whether HK-2 cells are capable to uptake the SGLT2-mediated glucose influx, HK-2 cells, identified by selectively and highly expressed SGLT2 as previous reported , and the fluorescent glucose were applied for glucose measurement in our assay.

For capturing the best readout for measuring SGLT2-mediated glucose uptake in HK-2 cells, we screen SGLT2 expressions during cell culturing up to passage 18. As shown in Fig. 1a, the SGLT2 expressions were constantly stable from passage 6 to passage 12, but its expression was significantly decreased at passage 18 , so the HK-2 cells used in current experiments were all finished before the passage 12.

Fig. 1

Endogenous SGLT2 of HK-2 cells mediates glucose uptake. a Western blot of SGLT2 expressions in serial passages of HK-2 cells. b HK-2 cells were incubated in Na+ or Na+-free buffer for 30 min with 200 M 2-NBDG. Both the bright field images and the fluorescent images were taken at10× magnification of objective. Na+, sodium buffer Na+, sodium free buffer. Scale bar, 100 m. c Quantification of 2-NBDG fluorescence intensity. The 2-NBDG values were normalized to the fluorescence intensity of Na+ group. Experiments were performed in triplicate and data are presented as mean±S.E.M. of three independent experiments. A.U., arbitrary units. The significance was determined by two-tailed paired t test group)

Single Cell Glucose Uptake Assays: A Cautionary Tale

1 Division of Cell Signalling and Immunology, School of Life Sciences, University of Dundee, Dundee, DD1 4HN, UK

2 Molecular Cell Physiology, Université Libre de Bruxelles , IBMM , Gosselies 6041, Belgium

* Correspondence: Linda V. Sinclair, Doreen A. Cantrell.

Received: 15 May 2020 Accepted: 10 August 2020 Published: 17 August 2020

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Why Measure Glucose Uptake

We can learn several things from measuring glucose uptake. Changes in glucose uptake can reflect overall changes in metabolism, but there are many specific processes as well. In cancer cells, measuring glucose uptake can monitor the overexpression of glucose transporters or identify glucose transporter inhibitors. With fat and muscle cells, changes in GLUT4 translocation upon insulin stimulation can be observed by measuring glucose uptake. In immunologically relevant cells, measuring glucose uptake can be used to follow the transformation of certain cell types from one stage to another.

System

Monitor GLUT4 translocation in response to insulin

Immunologically relevant cells

Any cancer cell

Monitor glucose transporter overexpression, identify glucose transporter inhibitors or measure general changes in metabolism

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