J. Biochem. Biophys.

Methods 64 (2005) 207 – 215

www.elsevier.com/locate/jbbm

2-NBDG as a fluorescent indicator for direct

glucose uptake measurement
Chenhui Zou a, Yajie Wang b, Zhufang Shen a,*
a
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College,
1 Xiannongtan Street, Beijing 100050, China
b
Center for Laboratory Diagnosis, Beijing Tiantan Hospital Affiliated Capital University of Medical Sciences,
6 Tiantan Xili, Beijing 100050, China
Received 6 January 2005; received in revised form 24 May 2005; accepted 8 August 2005

Abstract

Evaluation of glucose uptake ability in cells plays a fundamental role in diabetes mellitus
research. In this study, we describe a sensitive and non-radioactive assay for direct and rapid
measuring glucose uptake in single, living cells. The assay is based on direct incubation of
mammalian cells with a fluorescent d-glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)
amino]-2-deoxy-d-glucose (2-NBDG) followed by flow cytometric detection of fluorescence
produced by the cells. A series of experiments were conducted to define optimal conditions for this
assay. By this technique, it was found that insulin lost its physiological effects on cells in vitro
meanwhile some other anti-diabetic drugs facilitated the cell glucose uptake rates with mechanisms
which likely to be different from those of insulin or those that were generally accepted of each drug.
Our findings show that this technology has potential for applications in both medicine and research.
D 2005 Elsevier B.V. All rights reserved.

Keywords: Diabetes; 2-NBDG; Glucose uptake

* Corresponding author. Tel./fax: +86 10 83172669.

E-mail addresses: zch@imm.ac.cn (C. Zou), tiantanwyj@yahoo.com.cn (Y. Wang), shenzhf@imm.ac.cn
(Z. Shen).

0165-022X/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbbm.2005.08.001
208 C. Zou et al. / J. Biochem. Biophys. Methods 64 (2005) 207–215

1. Introduction

Diabetes mellitus is one of the most prevalent and serious metabolic diseases and the
principal cause of morbidity and mortality in the human. In diabetes, there is a failure to
increase glucose uptake into peripheral tissues in response to insulin, leading to chronically
elevated levels of glucose in the circulation [1]. A focus of current anti-diabetic medicine
research is the development and screening of compounds with potential insulinomimetic
effects to stimulate rate of cell glucose uptake [2]. Most studies on glucose uptake are
commonly carried out using radiotracers such as 2-deoxy-d-[14C]glucose or 2-deoxy-d-
[3H]glucose. However, there are several disadvantages associated with the radiotracer such
as disposal of radioactive waste or radioactive cleanup. More importantly, it cannot directly
measure glucose uptake in single, living cells [3]. We present an assay that is based on
direct incubation of mammalian cells with a fluorescent d-glucose analog 2-[N-(7-
nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) followed by flow
cytometric detection of fluorescence produced by the cells.
This is the first report, which combines a fluorescent glucose analog with flow
cytometry technique that allowing direct and more sensitive measurement of glucose
uptake cell by cell. In addition, there has been no method previously to directly acquire the
relative fluorescent density of each cell, and we solved this problem by introducing a free
software WinMDI to digitize flow cytometry plots for further statistical analysis. This
technique is characterized by its simplicity and accuracy and can be extended to wide
range of applications such as screening leading compounds for anti-diabetic drug
candidates or offering a detection method to help provide insights into the mechanisms of
glucose accumulation and metabolism in cells.

2. Materials and methods

2.1. Cell culture

HepG2 human hepatocarcinoma cells and L6 rat skeletal muscle cells were cultured in
MEM (Minimum Essential Medium) and a-MEM growth medium with 10% fetal bovine
serum (FBS), respectively. Cells were maintained at 37 8C in a humidified 5% CO2
environment.

2.2. Glucose uptake assay

Cells were plated at 1  104/well in 96-well plates and used at subconfluence after 24
h preincubation. The authors strongly recommended that experiments be performed within
48 h. Longer incubation time, excessively confluent or too few cells will reduce the
reproducibility of the assay. For experiments, all culture medium was removed from each
well and replaced with 100 Al of culture medium in the absence or presence of fluorescent
2-NBDG or 2-NBDG together with compounds at indicated concentrations. Plates were
incubated at 37 8C with 5% CO2 for a period of time as described in each experiment
before flow cytometry analysis.
 C. Zou et al. / J. Biochem. Biophys. Methods 64 (2005) 207–215 209

2.3. Flow cytometry

The 2-NBDG uptake reaction was stopped by removing the incubation medium and
washing the cells twice with pre-cold phosphate buffered saline (PBS). Cells in each well
were subsequently resuspended in 200 Al pre-cold fresh growth medium and then given a
final Propidium Iodide (PI) concentration of 1 Ag/ml and maintained at 4 8C for later flow
cytometry analysis performed within 30 min. For each measurement, data from 2000
single cell events was collected using a FACScalibur (Becton Dickinson Immunocyto-
metry Systems, SanJose, CA) flow cytometer within 20 s.

2.4. Digitization of flow cytometry events

Data from each flow cytometric measurement was stored as a listmode file, which
refers to a correlated data file where each event was listed sequentially. A listmode file
provided at least 7 parameters of each cell event, only 4 of them were chosen for statistical
analysis: relative size (Forward Scatter-FSC), granularity or internal complexity (Side
Scatter-SSC), FL1 and FL3 fluorescence intensity (2-NBDG and PI, respectively) of cells.
However, listmode files are created in FCS2.0 (Flow Cytometry Standard) format and
cannot be opened directly by data analysis software such as SPSS. We used free software
WinMDI version 2.9 (Scripps Research Institute) to convert FCS formatted files to text
files with all parameters of cell events, which can be directly imported by data analysis
software. Relative scatter light or fluorescence intensity of each event was assigned with
numbers ranging from 0 to 1023.

2.5. Statistical analysis

We can almost get unlimited cytometric events for data analysis, but too large sample
size will even make within-group differences significant, this kind of significance is
meaningless in practice. So, there is a need to determine a maximal sample size to ensure
that there is no significant within-group difference. In our experiments, n = 100. Since
cytometric events were listed sequentially, the first 100 of the total 2000 events were
chosen. We checked the normality of the data and they are not normal distribution.
Therefore, data are presented as medians. Statistical analysis was performed using
nonparametric Mann–Whitney test for identification of within-and between-group
differences. Significance was set a priori at P b 0.05. Calculations were run in SPSS for
windows version 11.5.

3. Results and discussion

2-NBDG (Molecular Probes) is a new fluorescent derivative of glucose modified with a

2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino group at the C-2 position (MW = 342.26)
[4]. This product showed intense fluorescence at 542 nm when excited at 467 nm.
Propidium Iodide (PI) is a nuclear dye known to permeate the cell membrane of dead cells
while living cell is resistant to this dye [5]. Abnormal changes of PI fluorescence intensity,
210 C. Zou et al. / J. Biochem. Biophys. Methods 64 (2005) 207–215

FSC (relative cell size or volume) or SSC (relative cell internal complexity) indicate
unusual physiological and/or toxicological effects due to incubation with 2-NBDG or
other compounds.
Four parameters of each 2000 cell events were displayed with WinMDI 2D dot plots
showed the different patterns of cell size, granularity, and extent that cells stained with 2-
NBDG and PI fluorescent dye (Fig. 1). They were not necessarily the same for each cell.
Data of each specimen were obtained in two separate parallel experiments. In general, we
did not find significant change in FSC, SSC, or PI fluorescence intensity unless
particularly mentioned. The major concern of this study was the intensity of the
fluorescence resulted from different rates of 2-NBDG uptake by cells.
d-glucose is transported into the cell by the glucose transporter (GLUT). Previous
studies showed that 2-NBDG uptaken by several cell types was significantly inhibited in
the presence of d-glucose while l-glucose did not appear to have the same inhibition
effect, suggesting that 2-NBDG and d-glucose are competing for the glucose transporter
to enter the cell [6–8]. A study by Ball et al. (2002) showed that 2-NBDG
concentrations higher than 0.25 mM might have a high degree of self-quenching [2].
In order to optimize 2-NBDG concentration for glucose uptake measurement, cells were

Fig. 1. Flow cytometry dot plots of 2000 cells. Different patterns of 2D plots indicating the relative size
(Forward Scatter-FSC), relative granularity or internal complexity (Side Scatter-SSC) and relative FL1 and FL3
fluorescence intensity (2-NBDG and PI, respectively) of cells. (a, b) HepG2 human hepatocarcinoma cell. (c, d)
L6 rat skeletal muscle cell.
 C. Zou et al. / J. Biochem. Biophys. Methods 64 (2005) 207–215 211

Fig. 2. Fluorescence intensity change of 2-NBDG in cells. Cells were incubated with 0, 5, 10, 20, 40 AM of 2-NBDG
for 10 min, respectively. (a) HepG2 human hepatocarcinoma cell. (b) L6 rat skeletal muscle cell. *: P b 0.05, other
concentrations versus any one of 0 AM.

incubated with 0, 5, 10, 20, 40 AM of 2-NBDG for 10 min, respectively. The

experimental results showed that a fast 2-NBDG uptake by cells at concentration as low
as 5 AM yielded a significant signal-to-noise ratio (Fig. 2). 2-NBDG has not been used
to measure glucose uptake in HepG2 human hepatocarcinoma cells and L6 rat skeletal
muscle cells previously. However, a concentration of 10 AM was taken from the
literature, which gave reproducible and reliable experimental results [2]. Based on our
experimental results and previous publications, the concentration of 2-NBDG was
chosen as 10 AM.
Optimal staining time was determined with 10 AM 2-NBDG incubations at 37 8C for 0,
15, 30, 60, 120 or 180 min. It was previously hypothesized that saturation of 2-NBDG
uptake would occur when it reached a dynamic equilibrium level of entry and
decomposition of 2-NBDG in cells, we found the rate of uptake was higher than that of
decomposition at least within the first 3 h. The highest rate of signal augment was
observed from 0 to 60 min (Fig. 3). An incubation time of 60 min was chosen as a
reasonable time for the analysis of the effects that drugs acting on cells.

Fig. 3. Changes in fluorescence after incubation for 0–180 min with 10 AM 2-NBDG. (a) HepG2 human
hepatocarcinoma cell. (b) L6 rat skeletal muscle cell. *: P b 0.05, other incubation time versus any one of 0 min.
212 C. Zou et al. / J. Biochem. Biophys. Methods 64 (2005) 207–215

Based on the facts that when 2-NBDG was incorporated in cells, it was phosphorylated
at the C-6 position, and then this 2-NBDG metabolite was readily decomposed to a non-
fluorescent derivative [7], the mechanisms of this process remained unelucidated. So, the
fluorescence intensity should reflect a dynamic equilibrium level of generation and
decomposition of the derivative. Our results confirmed this hypothesis (Fig. 4). Without 2-
NBDG supplement, the fluorescence intensity in cells decreased gradually over time and
this process could be halted at low temperature. This could be partly explained by the
inhibition of metabolic enzyme activity by the cold, indicating that maintaining cells at 4
8C before flow cytometry measurements was an essential step for the accuracy of
experimental results. Fluorescence attenuation of 2-NBDG during experimental procedure
should be taken into consideration. In addition, cell suspension stored at 37 8C for a certain
period of time also displayed reduced cell volume (data not shown) while low temperature
helped maintain cell configuration.
Cells received 1 h exposure to 10 AM 2-NBDG for 1 h as control or 2-NBDG together
with five anti-diabetic drugs or candidates: Insulin, Rosiglitazone, Glibenclamide,
Metformin and Chiglitazar in 3 concentrations as indicated in Fig. 5. Only one of the
medians of two separate parallel experiments was presented owing to limited space. Effects
were registered as an increase in the dose-dependent fluorescence. Rosiglitazone
moderately, while Glibenclamide and Chiglitazar significantly enhanced the rates of 2-
NBDG uptake at higher concentrations (10 5M, 10 6M) in HepG2 cells, while L6 cells
was only response to Glibenclamide at 10 5M, reflecting different molecular mechanisms
that trigger glucose uptake involved in these two cell lines. Fluorescence augment at other
concentrations of drugs was indistinguishable from the control. Interestingly, we noticed
that insulin lost its physiological effects on cells in vitro while Glibenclamide, an inhibitor
of the ATP-sensitive potassium channel which causes depolarization of the h-cell
membrane and then triggers the opening of voltage-gated Ca2+ channels, subsequently
elicits a rise in intracellular Ca2+ which stimulates the exocytosis of insulin-containing
secretary granules [9]; Rosiglitazone, which improves insulin-stimulated glucose uptake by

Fig. 4. Inhibitory effects of intracellular 2-NBDG metabolism by low temperature. Cells were incubated in culture
medium with 10 AM 2-NBDG for 1 h, and then resuspended in culture medium without 2-NBDG. Cells were
subsequently placed in different conditions, first at 37 8C and then at 4 8C with time as indicated in the figure. (a)
HepG2 human hepatocarcinoma cell. (b) L6 rat skeletal muscle cell. *: P b 0.05 other samples versus any one that
incubated at 4 8C for 2.5 h.
 C. Zou et al. / J. Biochem. Biophys. Methods 64 (2005) 207–215 213

Fig. 5. Cells received 1h exposure to 10 AM 2-NBDG as control (con) or 2-NBDG together with five anti-diabetic
drugs or candidates, respectively: Insulin (ins), Rosiglitazone (ros), Glibenclamide (gli), Metformin (met),
Chiglitazar (chi) in 3 concentrations as indicated in the figure. Results were the mean value of two separate
parallel experiments. (a) HepG2 human hepatocarcinoma cell. (b) L6 rat skeletal muscle cell. *: P b 0.05, drugs or
candidates versus control.

the activation of the nuclear peroxisome proliferator-activated receptor g (PPARg)[10];

Chiglitazar, a patented candidate which is supposed to act as a novel PPARa/g dual
agonist, directly facilitated the rates of cell glucose uptake with mechanisms apparently
unlikely to those of insulin or mentioned above. Further elucidation of these pathways will
be needed.
Despite all advantages this technique will bring with, a small fraction of candidates,
which can enter cells and have autofluorescence that sharing adjacent emission wavelength
with 2-NBDG when excited, cannot be detected by this protocol since results from these
candidates may be false positive. Compounds or extracts from nature products with
fluoresphore in molecular structure or having color in solution are highly suspect.
Autofluorescence detection of these kinds of candidates is always essential.

4. Simplified description of the method and its applications

Evaluation of the ability of glucose uptake by organisms is one of the main aspects of
diabetes research. We here describe a new system for the rapid and direct glucose uptake
214 C. Zou et al. / J. Biochem. Biophys. Methods 64 (2005) 207–215

measurement with a fluorescent d-glucose analog 2-NBDG. Such non-radioactive tags are
easier to visualize and do not pose a radiation hazard. Each single cell was considered as
an individual sample in our experiments, we did not require destruction of the cells for
each data point, and our experimental design was bbefore-and-afterQ [11]. Thus, the use of
2-NBDG to directly determine the distribution of single cell glucose uptake rates in a
population provided significant advantage over traditional indirect methods of inferring
population-averaged rates from time-course data of bulk glucose concentrations [12]. Flow
cytometry technique allows simultaneous monitoring changes of cell volume, internal
complexity and fluorescence of different dye at high sampling speed (100 cells/s), large
amount of samples (n = 2000) made experimental results highly reproducible. Further-
more, the technique using 2-NBDG as a fluorescent indicator is not restricted to screening
insulinomimetic compounds, it also provides insights into the mechanisms of glucose
accumulation and metabolism in unlimited cell types, primary or cell line, with other
proper experiment strategies. In conclusion, the application of 2-NBDG as a fluorescent
indicator provides a novel strategy not only for developing anti-diabetic drugs but also for
the fundamental research on diabetes.

Acknowledgments

The research was supported by the National Nature Science Foundation of China
(grants 90209037). The authors wish to thank professors S. Zhao, Z.Y. Song and Z.W. Hu
for the help with the manuscript.

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