ab136955
Glucose Uptake
Assay Kit
(Colorimetric)
Instructions for use:
For sensitive and accurate measurement of glucose
uptake in cells.
This product is for research use only and is not intended
for diagnostic use.
Version 11 Last Updated: 29 December 2015
Table of Contents
INTRODUCTION
1
1.
BACKGROUND
1
2.
ASSAY SUMMARY
2
GENERAL INFORMATION
3
3.
PRECAUTIONS
3
4.
STORAGE AND STABILITY
3
5.
LIMITATIONS
4
6.
MATERIALS SUPPLIED
4
7.
MATERIALS REQUIRED, NOT SUPPLIED
5
8.
TECHNICAL HINTS
6
ASSAY PREPARATION
7
9.
10.
REAGENT PREPARATION
STANDARD PREPARATION
7
9
ASSAY PROCEDURE
10
11.
10
ASSAY PROCEDURE
DATA ANALYSIS
14
12.
CALCULATIONS
14
13.
TYPICAL DATA
15
RESOURCES
16
14.
TROUBLESHOOTING
16
15.
FAQS
18
16.
NOTES
19
INTRODUCTION
INTRODUCTION
1. BACKGROUND
Glucose Uptake Assay Kit (Colorimetric) (ab136955) is an assay that
uses the glucose analog 2-deoxyglucose (2-DG) to detect and quantify
glycose uptake in cells. This easy to use, non-radioactive kit is highly
sensitive and can detect as low as 10 pmol 2-DG uptake per well.
Among many different methods available for measuring glucose uptake,
2-deoxyglucose (2-DG) has been widely used because of its structural
similarity to glucose. Similarly to glucose, 2-DG can be taken up by
glucose transporters and metabolized to 2-DG-6-phosphate (2-DG6P).
However, 2-DG6P cannot be metabolized further and thus accumulates
in the cells. The accumulated 2-DG6P is oxidized to generate NADPH,
which results in oxidation of a substrate. The oxidized substrate can then
be detected at OD=412 nm. The amount of accumulated NAPDH, and
therefore 2-DG6P, is directly proportional to 2-DG (or glucose) taken up
by cells.
Figure 1: Assay Procedure. 2-DG oxidation generates NADPH (step A),
which leads to production of an oxidized product that can be detected at OD
412 nm.
Glucose uptake is an important biological process for studying cell
signaling and glucose metabolism. This product can be used for the
measurement of glucose uptake in response to insulin, growth factors,
cytokines, mitogens or nutrients, as well as to analyze glucose
metabolism and signaling in various cell types and the screening of antidiabetic drugs.
ab136955 Glucose Uptake Assay Kit (Colorimetric)
1
INTRODUCTION
2. ASSAY SUMMARY
Grow cells (control and treated) in serum free medium
LEAVE O/N
Wash cells and incubate in KRPH/2% BSA for 40
minutes
Add insulin (or other stimulator) +/- inhibitor + Add 2-DG
to cells
Prepare 2-DG6P standard curve
Degradation of unused and endogenous NAD(P)
Recycling Amplification Reaction
Measure uptake at OD412 nm in a kinetic mode
ab136955 Glucose Uptake Assay Kit (Colorimetric)
2
GENERAL INFORMATION
GENERAL INFORMATION
3. PRECAUTIONS
Please read these instructions carefully prior to beginning the
assay.

All kit components have been formulated and quality control tested
to function successfully as a kit.

We understand that, occasionally, experimental protocols might
need to be modified to meet unique experimental circumstances.
However, we cannot guarantee the performance of the product
outside the conditions detailed in this protocol booklet.

Reagents should be treated as possible mutagens and should be
handle with care and disposed of properly. Please review the Safety
Datasheet (SDS) provided with the product for information on the
specific components.

Observe good laboratory practices. Gloves, lab coat, and protective
eyewear should always be worn. Never pipet by mouth. Do not eat,
drink or smoke in the laboratory areas.

All biological materials should be treated as potentially hazardous
and handled as such. They should be disposed of in accordance
with established safety procedures.
4. STORAGE AND STABILITY
Store kit at -20ºC in the dark immediately upon receipt. Kit has a
storage time of 1 year from receipt, providing components have not
been reconstituted.
Refer to list of materials supplied for storage conditions of individual
components. Observe the storage conditions for individual prepared
components in the Materials Supplied section.
Aliquot components in working volumes before storing at the
recommended temperature. Reconstituted components are stable for
2 months.
ab136955 Glucose Uptake Assay Kit (Colorimetric)
3
GENERAL INFORMATION
5. LIMITATIONS

Assay kit intended for research use only. Not for use in diagnostic
procedures.

Do not mix or substitute reagents or materials from other kit lots or
vendors. Kits are QC tested as a set of components and
performance cannot be guaranteed if utilized separately or
substituted.
6. MATERIALS SUPPLIED
Item
Amount
Assay Buffer
25 mL
Storage
Condition
(Before
Preparation)
-20°C
Extraction Buffer
17 mL
-20°C
-20°C
Neutralization Buffer
2.5 mL
-20°C
-20°C
Enzyme Mix (lyophilized)
1 vial
-20°C
-20°C
Glutathione Reductase
(lyophilized)
Recycling Mix (lyophilized)
2 vials
-20°C
-20°C
1 vial
-20°C
-20°C
Substrate-DTNB
(lyophilized)
2-Deoxyglucose (2-DG),
10 mM
2-DG6P standard
(lyophilized)
2 vials
-20°C
-20°C
1 mL
-20°C
-20°C
1 vial
-20°C
-20°C
ab136955 Glucose Uptake Assay Kit (Colorimetric)
Storage
Condition
(After
Preparation)
-20°C
4
GENERAL INFORMATION
7. MATERIALS REQUIRED, NOT SUPPLIED
These materials are not included in the kit, but will be required to
successfully perform this assay:

Microplate reader capable of measuring absorbance at OD 412 nm

MilliQ water or other type of double distilled water (ddH2O)

Pipettes and pipette tips, including multi-channel pipette

Assorted glassware for the preparation of reagents and buffer
solutions

Tubes for the preparation of reagents and buffer solutions

Sterile 96 well plate with clear flat bottom

Plate sealing tape

General tissue culture supplies

PBS

Krebs-Ringer-Phosphate-Hepes (KRPH) buffer: 20 mM HEPES,
5 mM KH2PO4, 1 mM MgSO4, 1 mM CaCl2, 136 mM NaCl, 4.7 mM
KCl, pH 7.4

BSA

Adipocyte culture medium (serum free and serum supplemented)
ab136955 Glucose Uptake Assay Kit (Colorimetric)
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GENERAL INFORMATION
8. TECHNICAL HINTS

This kit is sold based on number of tests. A ‘test’ simply refers
to a single assay well. The number of wells that contain sample,
control or standard will vary by product. Review the protocol
completely to confirm this kit meets your requirements. Please
contact our Technical Support staff with any questions.

Selected components in this kit are supplied in surplus amount to
account for additional dilutions, evaporation, or instrumentation
settings where higher volumes are required. They should be
disposed of in accordance with established safety procedures.

Avoid foaming
components.

Avoid cross contamination of samples or reagents by changing tips
between sample, standard and reagent additions.

Ensure plates are properly sealed or covered during incubation
steps.

Ensure all reagents and solutions are at the appropriate temperature
before starting the assay.

Samples which generate values that are greater than the most
concentrated standard should be further diluted in the appropriate
sample dilution buffer.

Make sure you have the right type of plate for your detection method
of choice.

Make sure all necessary equipment is switched on and set at the
appropriate temperature.
or
bubbles
when
ab136955 Glucose Uptake Assay Kit (Colorimetric)
mixing
or
reconstituting
6
ASSAY PREPARATION
ASSAY PREPARATION
9. REAGENT PREPARATION

9.1.
Briefly centrifuge small vials at low speed prior to opening
Assay Buffer:
Ready to use as supplied. Equilibrate to room temperature before
use. Store at -20°C.
9.2.
Extraction Buffer:
Ready to use as supplied. Equilibrate to room temperature before
use. Store at -20°C.
9.3.
Neutralization Buffer:
Ready to use as supplied. Equilibrate to room temperature before
use. Store at -20°C.
9.4.
Enzyme Mix:
Reconstitute with 220 µL Assay Buffer. Pipette up and down to
dissolve completely. Aliquot enzyme so that you have enough
volume to perform the desired number of assays. Store at -20°C.
Avoid repeated freeze/thaw cycles. Once reconstituted, use within
2 months.
9.5.
Glutathione Reductase:
Reconstitute with 1.1 mL Assay Buffer. Pipette up and down to
dissolve completely. Aliquot enzyme so that you have enough
volume to perform the desired number of assays. Store at -20°C.
Avoid repeated freeze/thaw cycles. Once reconstituted, use within
2 months.
9.6.
Recycling Mix:
Reconstitute with 220 µL Assay Buffer. Pipette up and down to
dissolve completely. Aliquot mix so that you have enough volume
to perform the desired number of assays. Store at -20°C. Avoid
repeated freeze/thaw cycles. Once reconstituted, use within 2
months.
ab136955 Glucose Uptake Assay Kit (Colorimetric)
7
ASSAY PREPARATION
9.7.
Substrate-DNTB:
Reconstitute with 1 mL Assay Buffer. Pipette up and down to
dissolve completely. Aliquot substrate so that you have enough
volume to perform the desired number of assays. Store at -20°C.
Once reconstituted, use within 2 months.
9.8.
2-Deoxyglucose (2-DG) (10 mM):
Ready to use as supplied. Equilibrate to room temperature before
use. Store at -20°C.
9.9.
2-DG6P Standard:
Reconstitute with 100 µL ddH2O to generate a 10 mM (10
nmol/µL) 2-DG6P solution. Pipette up and down to dissolve
completely. Aliquot standard so that you have enough volume to
perform the desired number of assays. Store at -20°C. Once
reconstituted, use within 2 months. Keep on ice while in use.
ab136955 Glucose Uptake Assay Kit (Colorimetric)
8
ASSAY PREPARATION
10. STANDARD PREPARATION

Always prepare a fresh set of standards for every use.

Diluted standard solution is unstable and must be prepared
immediately prior use. Do not store for future use.

Standard dilution can be prepared during insulin incubation step
(step 11.1.6, page 10)
10.1. Prepare 500 µL of 0.1 mM (100 pmol/µL) 2-DG6P Standard by
adding 5 µL of the 10 mM 2-DG6P Standard to 495 µL Assay
Buffer and mix well.
10.2. Prepare 500 µL of 0.01 mM (10 pmol/µL) by adding 50 µL of
0.1 mM 2-DG6P standard (section 10.1) to 450 µL Assay Buffer.
10.3. Using 0.01 mM standard, prepare standard curve dilution as
described in the table in a microplate or microcentrifuge tubes:
Standard #
Volume of
Standard
(µL)
Assay
Buffer
(µL)
End Conc
2-DG6P in well
(pmol/well)
150
Final
volume
standard in
well (µL)
50
1
0
2
3
6
12
144
138
50
50
20
40
4
5
18
24
132
126
50
50
60
80
6
30
120
50
100
0
Each dilution has enough amount of standard to set up duplicate
readings (2 x 50 µL).
ab136955 Glucose Uptake Assay Kit (Colorimetric)
9
ASSAY PROCEDURE
ASSAY PROCEDURE
11. ASSAY PROCEDURE

Equilibrate all materials and prepared reagents to correct
temperature prior to use.

We recommended to assay all standards, controls and samples
in duplicate.

Prepare all reagents, working standards, and samples as
directed in the previous sections.

This protocol has been optimized for 3T3-L1 adipocytes. For
other cell types, optimal incubation times and treatment
protocols may vary from these conditions and should be set by
the researcher. As initial recommendation, we suggest seeding
cells at a density of 60 – 80%.
11.1. Prepare Samples:
11.1.1.Seed cells at a density of ~ 1,500 3T3-L1 cells/well in
100 µL culture medium in a 96-well plate and differentiate
to mature adipocytes. Maintain differentiated cells for
another 4 days prior to use.
NOTE: prepare enough cells to be able to set up control
(untreated) and treated cells.
11.1.2.Wash adipocytes twice with PBS and starve in 100 µL
serum free adipocyte medium overnight to increase
glucose uptake.
11.1.3.Next day, wash cells 3X with PBS.
11.1.4.Starve cells for glucose by pre-incubating them with 100 µL
KRPH/2% BSA buffer for 40 minutes. Aspirate buffer.
11.1.5.Control (untreated) cells: Wash cells 3X with PBS.
Proceed to step 11.2.
11.1.6.Treated cells: Stimulate cells with medium (no insulin) or
with 1 µM insulin for 20 minutes to activate glucose
transporter. Total volume well = 100 µL. NOTE: this
incubation provides a quick and robust stimulation of
glucose uptake via GLUT transporters.
ab136955 Glucose Uptake Assay Kit (Colorimetric)
10
ASSAY PROCEDURE
11.1.7.Add 10 µL of 10 mM 2-DG (section 9.8) to cells, mix by
pipetting up and down.
11.1.8.Incubate cells for 20 minutes.
11.1.9.Wash cells 3X with PBS to remove exogenous 2-DG.
11.2. NAD(P) Degradation:
11.2.1.To degrade any unused NAD(P) left in the cells, lyse
control and treated cells by adding 80 µL Extraction Buffer
and pipetting up and down to release cells. NOTE: if using
a bigger culture surface, you can do this step with a cell
scraper.
NOTE: at this point, samples can be transferred to
microcentrifuge tubes for ease of handling. They can also be left
on the microplate if preferred.
11.2.2.Freeze/thaw once and heat samples at 85°C for 40
minutes to degrade endogenous NAD(P) and to denature
enzymes present in the sample. NOTE: if you have
covered your plate with film or lid, you might find some
condensation has formed. Spin the plate briefly (~ 500 rpm
1 minute) to get rid of the condensation bubbles before
you take the lid or the cover off.
11.2.3.Cool the cell lysates on ice for 5 minutes and neutralize
sample by adding 10 µL of Neutralization Buffer. Briefly
spin the samples to ensure proper mixing of reagents (~
500 rpm 1 – 2 minutes, or using soft spin cycle if
available).
11.2.4.Transfer supernatant to new tubes.
NOTE: pausing in the middle of the assay is not recommended.
However, if really necessary, we suggest you freeze your samples
at -80°C at this step as all proteins have been removed. Thaw
samples on ice when you are ready to proceed with the assay.
ab136955 Glucose Uptake Assay Kit (Colorimetric)
11
ASSAY PROCEDURE
11.3. Set up Reaction wells:
Set up reaction in a new 96 well clear plate with flat bottom.
Initial recommendation: dilute samples 1/10 in Assay Buffer to a
total volume of 50 µL (5 µL samples + 45 µL Assay Buffer).
We recommend performing several different sample dilutions with
Assay Buffer to ensure the readings fall within the standard curve.
You can use 1 – 50 µL sample/well (adjust to 50 µL/well with
Assay Buffer).
11.4. Reaction Mix:
11.4.1.Prepare 10 µL of Reaction Mix A (NADPH generation) for
each reaction. Mix enough reagents for the number of
assays (samples and controls) to be performed. Prepare a
master mix of the Reaction Mix to ensure consistency. We
recommend the following calculation: X µL component x
(Number reactions +1).
Component
Reaction Mix A (µL)
Assay Buffer
8
Enzyme Mix
2
11.4.2.Add 10 µL Reaction Mix A to each well (standard, control
and treated samples) and mix well by pipetting up and
down.
11.4.3.Incubate at 37°C for 1 hour.
11.4.4.Add 90 µL Extraction Buffer to each well, seal the
microplate with a sealing tape and heat it at 85°C for 40
minutes to degrade any unused NADP left in the sample.
11.4.5.Cool plate on ice for 5 minutes and neutralize reaction by
adding 12 µL of Neutralization Buffer.
ab136955 Glucose Uptake Assay Kit (Colorimetric)
12
ASSAY PROCEDURE
11.4.6.Prepare 38 µL of Reaction Mix B (recycling amplification
reaction) for each reaction. Mix enough reagents for the
number of assays (samples and controls) to be performed.
Prepare a master mix of the Reaction Mix to ensure
consistency. We recommend the following calculation: X
µL component x (Number reactions +1).
Component
Reaction Mix B (µL)
Glutathione Reductase
20
Substrate-DTNB
16
Recycling Mix
2
11.4.7.Add 38 µL Reaction Mix B to each well (standard, control
and treated cells) and mix well by pipetting up and down.
11.5. Detection:
11.5.1.Measure output at OD 412 nm on a microplate reader in a
kinetic mode, every 2 – 3 minutes, at 37°C protected from
light, until Standard #6 (100 pmol/well) reaches 1.5 -2 OD
value.
11.5.2.Take an additional endpoint reading of all samples and
standards.
ab136955 Glucose Uptake Assay Kit (Colorimetric)
13
DATA ANALYSIS
DATA ANALYSIS
12. CALCULATIONS

Samples producing signals greater than that of the highest standard
should be further diluted in appropriate buffer and reanalyzed, then
multiply the concentration found by the appropriate dilution factor.
12.1. Average the duplicate reading for each standard and sample.
12.2. Subtract the sample background control (cells not treated with
insulin nor 2-DG) from sample reading.
12.3. Subtract the mean absorbance value of the blank (Standard #1)
from all standard and sample readings. This is the corrected
absorbance.
12.4. Plot the corrected absorbance values for each standard as a
function of the final concentration of 2-DG6P.
12.5. Draw the best smooth curve through these points to construct the
standard curve. Calculate the trend line equation based on your
standard curve data (use the equation that provides the most
accurate fit).
12.6. Concentration of 2-DG in the test samples is calculated as:
2 ‒ 𝐷𝐺 𝑢𝑝𝑡𝑎𝑘𝑒 = 𝐵
𝑉 ∗ 𝐷 = 𝑝𝑚𝑜𝑙/µ𝐿 = 𝑛𝑚𝑜𝑙/𝑚𝐿 = µ𝑀
()
Where:
B = Amount of 2-DG6P in sample calculated from the standard
curve (pmol).
V = Original sample volume added into the reaction well (µL).
D = Sample dilution factor.
ab136955 Glucose Uptake Assay Kit (Colorimetric)
14
DATA ANALYSIS
13. TYPICAL DATA
TYPICAL STANDARD CURVE – Data provided for demonstration
purposes only. A new standard curve must be generated for each
assay performed
Figure 2: Standard calibration curve (a) and 2-DG uptake in a variety of cell
lines [(b), 3T3-L1 cells; (c), human adipocytes and (d), HeLa]. I = insulin; P =
phloretin (glucose transport inhibitor).
ab136955 Glucose Uptake Assay Kit (Colorimetric)
15
RESOURCES
RESOURCES
14. TROUBLESHOOTING
Problem
Assay not
working
Sample with
erratic
readings
Lower/ Higher
readings in
samples and
standards
Cause
Solution
Use of ice-cold buffer
Buffers must be at room
temperature
Plate read at
incorrect wavelength
Check the wavelength and filter
settings of instrument
Use of a different 96well plate
Colorimetric: Clear plates
Fluorometric: black wells/clear
bottom plate
Samples not
deproteinized (if
indicated on protocol)
Use provided protocol for
deproteinization
Cells/tissue samples
not homogenized
completely
Use Dounce homogenizer,
increase number of strokes
Samples used after
multiple free/ thaw
cycles
Aliquot and freeze samples if
needed to use multiple times
Use of old or
inappropriately
stored samples
Use fresh samples or store at 80°C (after snap freeze in liquid
nitrogen) till use
Presence of
interfering substance
in the sample
Check protocol for interfering
substances; deproteinize
samples
Improperly thawed
components
Thaw all components completely
and mix gently before use
Allowing reagents to
sit for extended times
on ice
Always thaw and prepare fresh
reaction mix before use
Incorrect incubation
times or
temperatures
Verify correct incubation times
and temperatures in protocol
Standard stock is at
incorrect
concentration
Always refer to dilutions
described in the protocol
ab136955 Glucose Uptake Assay Kit (Colorimetric)
16
RESOURCES
Problem
Standard
readings do
not follow a
linear pattern
Unanticipated
results
Cause
Pipetting errors in
standard or reaction
mix
Air bubbles formed in
well
Standard stock is at
incorrect
concentration
Measured at incorrect
wavelength
Samples contain
interfering
substances
Sample readings
above/ below the
linear range
ab136955 Glucose Uptake Assay Kit (Colorimetric)
Solution
Avoid pipetting small volumes
(< 5 µL) and prepare a master mix
whenever possible
Pipette gently against the wall of
the tubes
Always refer to dilutions described
in the protocol
Check equipment and filter setting
Troubleshoot if it interferes with
the kit
Concentrate/ Dilute sample so it is
within the linear range
17
RESOURCES
15. FAQs
Can I start working on a 3.5 cm petri dish and then continue the
assay in a 96-well plate after the lysis step?
Yes, you can. You have to increase the volumes for all reagents used to
ensure all cells are covered and not exposed to the air to dry out. Use
the same amount of reagents that you would to culture cells – for
example, for the serum starvation incubation, instead of using 100 µL
media for covering cells in the 96 well plate, you could use about 2 mL
media for a 3.5 cm plate.
What is the purpose of starvation with serum free medium if it is
then followed by starvation with glucose free buffer?
Serum starvation is required because serum can provide a potential
carbon source from which cells can then make glucose.
I need to culture my cells with 1% insulin. Should the insulin be
removed during either serum starvation step and/or the glucose
starvation stage?
There should be no insulin in the media during starvation. After
starvation, cells are stimulated with insulin to induce uptake of glucose
via the GLUT transporters.
ab136955 Glucose Uptake Assay Kit (Colorimetric)
18
RESOURCES
16. NOTES
ab136955 Glucose Uptake Assay Kit (Colorimetric)
19
RESOURCES
ab136955 Glucose Uptake Assay Kit (Colorimetric)
20
RESOURCES
ab136955 Glucose Uptake Assay Kit (Colorimetric)
21
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