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Amplite® Fluorimetric Glucose Quantitation Kit

Glucose dose response was measured with Amplite® Fluorimetric Glucose Quantitation Kit on a 96-well black plate using a Gemini microplate reader (Molecular Devices).
Glucose dose response was measured with Amplite® Fluorimetric Glucose Quantitation Kit on a 96-well black plate using a Gemini microplate reader (Molecular Devices).
Glucose dose response was measured with Amplite® Fluorimetric Glucose Quantitation Kit on a 96-well black plate using a Gemini microplate reader (Molecular Devices).
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Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Spectral properties
Excitation (nm)571
Emission (nm)584
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Excitation (nm)
571
Emission (nm)
584
Glucose, a monosaccharide, is the most important carbohydrate in biology. It is a source of energy and metabolic intermediate for cell growth. Glucose is one of the main products of photosynthesis and starts cellular respiration in both prokaryotes and eukaryotes. Glucose level is a key diagnostic parameter for many metabolic disorders. This glucose assay kit provides a quick and sensitive method for the measurement of glucose in various biological samples (e.g., serum, plasma, body fluid, food, growth medium, etc.). The kit uses our Amplite®Red substrate that making the kit recordable in a dual more, either fluorimetric or colorimetric readout. The kit provides all the essential components with an optimized assay protocol. The assay is robust, and can be readily adapted for high-throughput assays in a wide variety of applications that require the measurement of glucose. For example, the assay might be suitable for monitoring glucose level during fermentation and glucose feeding in protein expression processes. It might also be used for monitoring glucose transporters.

Platform


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black

Components


Example protocol


AT A GLANCE

Protocol summary

  1. Prepare and add Glucose standards and/or test samples (50 µL)
  2. Prepare and add Glucose Assay working solution (50 µL)
  3. Incubate at 37°C for 10 - 30 minutes
  4. Monitor fluroscence intensity at Ex/Em = 540/590 nm

Important notes
Thaw all the kit components at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Amplite™ Red stock solution (250X):
Add 100 µL of DMSO (Component E) into the vial of Amplite™ Red substrate (Component A). The stock solution should be used promptly. Any remaining solution should be aliquoted and refrozen at -20 oC. Note:  Avoid repeated freeze-thaw cycles. Note: The Amplite™ Red substrate is unstable in the presence of thiols such as dithiothreitol (DTT) and 2-mercaptoethanol. The final concentration of DTT or 2-mercaptoethanol in the reaction should be no higher than 10 µM. The Amplite™ Red substrate is also unstable at high pH (> 8.5). Therefore, the reaction should be performed at pH 7–8. The provided assay buffer (pH 7.4) is recommended.

2. Horseradish Peroxidase (HRP) stock solution (10 U/mL):
Add 1 mL of Assay Buffer (Component B) into the vial of Horseradish Peroxidase (Component C). Note: The unused HRP solution should be divided into single use aliquotes and stored at -20 oC.

3. Glucose Oxidase solution (100 U/mL):
Add 1 mL of Assay Buffer (Component B) into the vial of Glucose Oxidase (Component D). Note: The unused Glucose Oxidase solution should be divided into single use aliquotes and stored at -20 oC.

4. Glucose stock solution (800mM):
Add 1 mL of Assay Buffer (Component B) into the vial of Glucose (Component F). Note: The unused Glucose solution should be divided into single use aliquotes and stored at -20 oC.

PREPARATION OF STANDARD SOLUTION

glucose standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/40005

Prepare a glucose standard by diluting the appropriate amount of the 800 mM glucose stock solution into Assay Buffer (Component B) to produce glucose concentrations of 30 µM. Then perform 1:3 serial dilutions in Assay Buffer (Component B) to get approximately 10, 3, 1, 0.3, 0.1 and 0.03 µM serially diluted glucose standards. A non-glucose buffer control is included as blank control.

PREPARATION OF WORKING SOLUTION

Table 1. Assay working solution for one clear bottom 96-well microplate (2X)

Components

Volume

Amplite™ Red Stock Solution (250x)

20 μL

HRP Stock Solution (10 U/mL)

100 μL

Glucose Oxidase Solution (100 U/mL)

100 μL

Assay Buffer 

4.78 mL

Total volume

5 mL

SAMPLE EXPERIMENTAL PROTOCOL

Table 2. Layout of Glucose standards and test samples in a solid black 96-well microplate. GS = Glucose standard (GS1-GS7); BL = blank control; TS = test sample.

BLBLTSTS
GS1GS1......
GS2GS2......
GS3GS3  
GS4GS4  
GS5GS5  
GS6GS6  
GS7GS7  

Table 3. Reagent composition for each well

Glucose StandardBlank ControlTest Sample
Serial Dilutions: 50 µLAssay Buffer (Compound B): 50 µL50 µL

Note: High Concentration of glucose (e.g. 100 µM in test sample or standard) may cause reduced fluoroscence signal due to the overoxidation of Amplite™ red substrate (to a non-fluorescent product).

Glucose assay

  1. Add glucose standards and glucose containing test samples into a 96-well solid black microplate as described in Tables 2 and 3.

  2. Add 50 µL of Glucose Assay working solution into each well of glucose standard, blank control, and test samples (Table 2) to make the total glucose assay volume of 100 µL/well. Note: For a 384-well plate, add 25 µL of sample and 25 µL of assay reaction mixture into each well.

  3. Incubate the reaction for 10 to 30 minutes at 37 oC, protected from light.

  4. Monitor the fluorescence intensity with a fluorescence plate reader at Ex/Em= 530-570 nm/590-600 nm (optimal Ex/Em = 540/590 nm).

Spectrum


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spectrum

Spectral properties

Excitation (nm)571
Emission (nm)584

Images


Citations


View all 19 citations: Citation Explorer
Molecular programming of the hepatic lipid metabolism via a parental high carbohydrate and low protein diet in rainbow trout
Authors: Callet, Th{\'e}r{\`e}se and Li, Hongyan and Heraud, C{\'e}cile and Larroquet, Laurence and Lanuque, Anthony and Sandres, Franck and Terrier, Fr{\'e}d{\'e}ric and Surget, Anne and Corraze, Genevi{\`e}ve and Panserat, St{\'e}phane and others,
Journal: animal (2022): 100670
RIP140 inhibits glycolysis-dependent proliferation of breast cancer cells by regulating GLUT3 expression through transcriptional crosstalk between hypoxia induced factor and p53
Authors: Jacquier, Valentin and Gitenay, Delphine and Fritsch, Samuel and Bonnet, Sandrine and Gy{\H{o}}rffy, Bal{\'a}zs and Jalaguier, St{\'e}phan and Linares, Laetitia K and Cavaill{\`e}s, Vincent and Teyssier, Catherine
Journal: Cellular and Molecular Life Sciences (2022): 1--17
No adverse effect of a maternal high carbohydrate diet on their offspring, in rainbow trout (Oncorhynchus mykiss)
Authors: Callet, Therese and Li, Hongyan and Surget, Anne and Terrier, Frederic and Sandres, Franck and Lanuque, Anthony and Panserat, Stephane and Marandel, Lucie
Journal: PeerJ (2021): e12102
RIP140 inhibits glycolysis-dependent proliferation of cancer cells by regulating transcriptional crosstalk between hypoxia induced factor and p53
Authors: Jacquier, Valentin and Gitenay, Delphine and Fritsch, Samuel and Linares, Laetitia K and Bonnet, Sandrine and Jalaguier, St{\'e}phan and Cavaill{\`e}s, Vincent and Teyssier, Catherine
Journal: bioRxiv (2020)
Exploring the impact of a low-protein high-carbohydrate diet in mature broodstock of a glucose-intolerant teleost, the rainbow trout
Authors: Callet, Th{\'e}r{\`e}se and Hu, Huihua and Larroquet, Laurence and Surget, Anne and Liu, Jingwei and Plagnes-Juan, Elisabeth and Maunas, Patrick and Turonnet, Nicolas and Mennigen, Jan Alexander and Bobe, Julien and others,
Journal: Frontiers in physiology (2020): 303
One pathway is not enough: the cabbage stem flea beetle Psylliodes chrysocephala uses multiple strategies to overcome the glucosinolate-myrosinase defense in its host plants
Authors: Beran, Franziska and Sporer, Theresa and Paetz, Christian and Ahn, Seung-Joon and Betzin, Franziska and Kunert, Grit and Shekhov, Anton and Vass{\~a}o, Daniel G and Bartram, Stefan and Lorenz, Sybille and others,
Journal: Frontiers in plant science (2018): 1754
Hepatic glucose metabolic responses to digestible dietary carbohydrates in two isogenic lines of rainbow trout
Authors: Song, Xuerong and Marandel, Lucie and Dupont-Nivet, Mathilde and Quillet, Edwige and Geurden, Inge and Panserat, Stephane
Journal: Biology open (2018): bio032896
Glucose metabolism ontogenesis in rainbow trout (Oncorhynchus mykiss) in the light of the recently sequenced genome: new tools for intermediary metabolism programming
Authors: Mar, undefined and el, Lucie and Véron, Vincent and Surget, Anne and Plagnes-Juan, Elisabeth and Panserat, Stéphane
Journal: Journal of Experimental Biology (2016): 734--743
MicroRNAs regulate gene plasticity during cold shock in zebrafish larvae
Authors: Hung, I-Chen and Hsiao, Yu-Chuan and Sun, H Sunny and Chen, Tsung-Ming and Lee, Shyh-Jye
Journal: BMC genomics (2016): 922

References


View all 21 references: Citation Explorer
Insulin and glucose mediate opposite intracellular ionized magnesium variations in human lymphocytes
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Recombinant human glucose-6-phosphate dehydrogenase. Evidence for a rapid-equilibrium random-order mechanism
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Journal: Eur J Biochem (2002): 3417
Fluorescent microplate cell assay to measure uptake and metabolism of glucose in normal human lung fibroblasts
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Journal: Toxicol In Vitro (2002): 267
Glucose-induced alterations of intracellular ionized magnesium in human lymphocytes
Authors: Delva P, Degan M, Pastori C, Faccini G, Lechi A.
Journal: Life Sci (2002): 2119
Plasma glycohydrolase levels in patients with type 1 diabetes at onset and in subjects undergoing an intravenous glucose tolerance test
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Journal: Metabolism (2000): 1352
High glucose induces enhanced monocyte adhesion to valvular endothelial cells via a mechanism involving ICAM-1, VCAM-1 and CD18
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Journal: Endothelium (1999): 315
Enalaprilat inhibits hydrogen peroxide production by murine mesangial cells exposed to high glucose concentrations
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Journal: Nephrol Dial Transplant (1997): 456
Glucose transport, phosphorylation, and utilization in isolated porcine pancreatic islets
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Journal: Metabolism (1995): 261
Plasma oxidizability in subjects with normal glucose tolerance, impaired glucose tolerance, and NIDDM
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