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1. Product Manual Glucose Assay Kit Fluorometric Catalog Number STA 681 500 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Researc Creating Solutions for Life Science Research Introduction Glucose is a sugar used as an important source of energy in plants prokaryotes and eukaryotes via processes such as respiration and fermentation In plants algae and cyanobacteria the energy of light is synthesized into the storage form of sugars such as glucose More specifically in a downstream process known as the Calvin cycle carbon dioxide is incorporated into organic carbon compounds like ribulose bisphosphate Using ATP and NADPH from upstream light dependent reactions the resulting compounds are then reduced and removed to form further carbohydrates such as glucose In animals through the process of glycolysis followed by the citric acid cycle glucose is broken down to water and CO resulting in energy from ATP formation Glucose is often stored as a polymer such as glycogen In humans glucose is commonly measured in blood samples Bloodstream levels of glucose are normally under tight regulation Table 1 however high levels measured in fasting individuals may indicate prediabetes or diabetes Blood Glucose Range Table 1 Range of blood glucose levels in common animals Ref 1 Cell Biolabs Glucose Assay Kit is a simple fluorometric assay2. be used to interpret or calculate actual sample results 0 20 40 60 80 100 120 Glucose uM Figure 2 Glucose standard curve gt CELL BIOLABS INC P 0 4 a lt 1 0 25 0 0625 0 Serum pL Figure 3 Glucose detection in human serum using the Glucose Assay Kit Fluorometric References 1 Kahn C M ed 2005 Merck Veterinary Manual 9th ed Whitehouse Station Merck amp Co 2 Tatyana V et al Neurochem 2001 79 266 3 Aranoff S L Berkowitz K Shreiner B and Want L 2004 Diabet Spectrum 17 183 190 4 McMillan J M 1990 Clin Methods 3 Ed The History Physical and Lab Examinations Ch 141 662 665 5 Alonso M D Lomako J Lomako W M and Whelan W J 1995 Faseb J 9 1126 1137 6 Shao J Wang Z Yang T Ying H Zhang Y and Liu S 2015 Int J Endocrinol 2015 1 9 Warranty These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used in accordance with their instructions THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSED WARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE CELL BIOLABS s sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of CELL BIOLABS to repair or replace the products In no event shall CELL BIOLABS be liable for any proximate incid
3. ental or consequential damages in connection with the products T7 Jau BIOLABS INC Contact Information Cell Biolabs Inc 7758 Arjons Drive San Diego CA 92126 Worldwide 1 858 271 6500 USA Toll Free 1 888 CBL 0505 E mail tech cellbiolabs com www cellbiolabs com 2015 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing eo CELL BIOLABS INC AN
4. in PBS or 1X Assay Buffer Homogenize or sonicate the cells on ice Centrifuge to remove debris Cell lysates may be assayed undiluted or diluted as necessary in 1X Assay Buffer Serum plasma or urine To remove insoluble particles centrifuge at 10 000 rpm for 5 min Dilute the supernatant from 1 50 to 1 200 with 1X Assay Buffer just prior to performing the assay Notes e All samples should be assayed immediately or stored at 80 C for up to 1 2 months Run proper controls as necessary Optimal experimental conditions for samples must be determined by the investigator Always run a standard curve with samples e Samples with NADH concentrations above 10 uM and glutathione concentrations above 50 uM will oxidize the Fluorometric Probe and could result in erroneous readings To minimize this interference it is recommended that superoxide dismutase SOD be added to the reaction at a final concentration of 40 U mL Tatyana et al Ref 2 e Avoid samples containing DTT or B mercaptoethanol since Resorufin is not stable in the presense of thiols above 10 uM Preparation of Standard Curve Prepare fresh Glucose standards before use by diluting in 1X Assay Buffer First dilute the stock Glucose Standard 400 mM solution 1 10 in 1X Assay Buffer for a 40 mM Glucose Solution e g add 5 uL of the stock 400 mM Glucose Standard to 45 uL of 1X Assay Buffer Use the 40 mM Glucose Solution to prepare a series of the remaining Glucose standards according
5. ingly Avoid multiple freeze thaw cycles Store the 10X Assay Buffer at room temperature Preparation of Reagents e 1X Assay Buffer Dilute the stock 10X Assay Buffer 1 10 with deionized water for a 1X solution Stir or vortex to homogeneity e Reaction Mix Prepare a Reaction Mix by diluting the Fluorometric Probe 1 100 HRP 1 500 and Glucose Oxidase 1 50 in 1X Assay Buffer For example add 50 uL Fluorometric Probe stock solution 10 uL HRP stock solution and 100 uL of Glucose Oxidase to 4 84 mL 1X Assay Buffer for a total of 5 mL The above example Reaction Mix volume is enough for 100 assays The Reaction Mix is stable for 1 day at 4 C Note Prepare only enough for immediate use by scaling the above example proportionally CELL BIOLABS INC yA o gt Preparation of Samples Cell culture supernatants Cell culture media formulated with glucose should be avoided To remove insoluble particles centrifuge at 10 000 rpm for 5 min The cell conditioned media may be assayed directly or diluted as necessary Prepare the Glucose standard curve in non conditioned media without glucose Note Maintain pH between 7 and 8 for optimal working conditions as the Fluorometric Probe is unstable at high pH gt 8 5 Tissue lysates Sonicate or homogenize tissue sample in cold PBS or 1X Assay Buffer and centrifuge at 10000 x g for 10 minutes at 4 C Perform dilutions in 1X Assay Buffer Cell lysates Resuspend cells at 1 2 x 10 cells mL
6. nosylhomocysteine SAH ELISA Combo Kit 7 STA 674 Glutamate Assay Kit 8 STA 675 Hydroxyproline Assay Kit 9 STA 680 Glucose Assay Kit Colorimetric 10 STA 682 Total Carbohydrate Assay Kit D Me we P CELL BIOLABS INC Kit Components Box 1 shipped at room temperature 1 Glucose Standard Part No 268001 One 500 uL tube at 400 mM 2 10X Assay Buffer Part No 268002 One 25 mL bottle 3 Fluorometric Probe Part No 268101 One 250 uL amber tube 4 HRP Part No 234402 One 100 uL tube at 100 U mL Box 2 shipped on blue ice packs 1 Glucose Oxidase Part No 268004 One 500 uL tube at 100U mL Note One unit is defined as the amount of enzyme that will oxidize 1 0 micromole of beta D glucose to D gluconic acid and hydrogen peroxide per minute at pH 5 1 at 35 C Materials Not Supplied 1 Distilled or deionized water 1X PBS 10 uL to 1000 uL adjustable single channel micropipettes with disposable tips 50 uL to 300 uL adjustable multichannel micropipette with disposable tips Standard 96 well fluorescence black microtiter plate and or black cell culture microplate Multichannel micropipette reservoir ae Se ee Fluorescence microplate reader capable of reading excitation in the 530 570 nm range and emission in the 590 600 nm range Storage Upon receipt store the Glucose Standard Fluorometric Probe HRP and Glucose Oxidase at 20 C The Fluorometric Probe is light sensitive and must be stored accord
7. that measures the amount of total glucose present in foods or biological samples in a 96 well microtiter plate format Each kit provides sufficient reagents to perform up to 500 assays including blanks glucose standards and unknown samples Sample glucose concentrations are determined by comparison with a known glucose standard The kit has a detection sensitivity limit of 1 56 uM glucose D CELL BIOLABS INC i Assay Principle Cell Biolabs Total Glucose Assay Kit measures total glucose within food or biological samples Glucose is oxidized by glucose oxidase into D gluconic acid plus hydrogen peroxide The hydrogen peroxide is then detected with a highly specific fluorometric probe Horseradish peroxidase catalyzes the reaction between the probe and hydrogen peroxide which bind in a 1 1 ratio Samples are compared to a known concentration of glucose standard within the 96 well microtiter plate format Samples and standards are incubated for 45 minutes and then read with a standard 96 well fluorometric plate reader Figure 1 Glucose O Glucose Fluorometric Probe Oxidase D Gluconic Acid Figure 1 Glucose assay principle Related Products 1 STA 398 Free Glycerol Assay Kit Colorimetric STA 399 Free Glycerol Assay Kit Fluorometric STA 670 Homocysteine ELISA Kit STA 671 S Adenosylhomocysteine SAH ELISA Kit STA 672 S Adenosylmethionine SAM ELISA Kit STA 671 C S Adenosylmethionine SAM ELISA and S Ade
8. to Table below 40 mM Glucose Glucose Standard Solution 1X Assay Buffer Glucose uM mg dL Tubes uL uL 1 4 1596 100 1 8 2 250 of Tube 1 250 50 0 9 3 250 of Tube 2 250 25 0 45 4 250 of Tube 3 250 125 0 225 5 250 of Tube 4 250 6 25 0 113 6 250 of Tube 5 250 3 13 0 056 7 250 of Tube 6 250 1 56 0 028 8 0 250 0 0 Table 2 Preparation of Glucose Standards CELL BIOLABS INC ean o gt Assay Protocol 1 Prepare and mix all reagents thoroughly before use Each sample including unknowns and standards should be assayed in duplicate or triplicate 2 Add 50 uL of each glucose standard or unknown sample into wells of a black microtiter plate suitable for a fluorescence plate reader 3 Add 50 uL of Reaction Mix to each well Mix the well contents thoroughly and incubate for 30 45 minutes at 37 C protected from light Note This assay is continuous not terminated and therefore may be measured at multiple time points to follow the reaction kinetics 4 Read the plate with a fluorescence microplate reader equipped for excitation in the 530 570 nm range and for emission in the 590 600 nm range 5 Calculate the concentration of glucose within samples by comparing the sample RFU to the standard curve Example of Results The following figures demonstrate typical Glucose Assay Kit Fluorometric results One should use the data below for reference only This data should not
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