UA-Glo®Glucose Luminescence Detection Kit

The UA-Glo® Glucose Luminescence Assay Kit detects glucose levels through a coupled reaction involving glucose dehydrogenase, reductase, and luciferase. Glucose dehydrogenase acts on glucose to reduce NADP to NADPH, while the reductase uses NADPH to convert its substrate (which is also a luciferin precursor) into luciferin. Finally, luciferin produces light upon action by luciferase, and this luminescence is detected; the intensity of the luminescence is directly proportional to the glucose concentration.

The UA-Glo® Glucose Luminescence Assay Kit is a homogeneous assay reagent that requires only a single addition for detection, thereby reducing experimental errors that may arise from multiple additions. This reagent features a high signal-to-noise ratio, excellent reproducibility, and good stability, making it suitable for batch processing of various samples, particularly for high-throughput screening of compounds.

UA-Glo®The components and specifications of the glucose luminescence assay kit are as follows. Detectable:96-The number of plate reactions is determined by the amount added to each well.50mu calLlifEnglish:Sample and50mu calLlifEnglish:Glucose reagent calculation.

Specifications	Component	96-Orifice plate reaction number
5ml	Fluorescein detection solution:5 mL Reductase substrate:25mu calLlifEnglish: Glucose(10 mM):50mu calLlifEnglish:	100 vehicles
50ml lotion	Fluorescein detection solution:50 mL of milk Reductase substrate:250mu calLlifEnglish: Glucose(10 mM):50mu calLlifEnglish:	1,000

UA-Glo®The glucose luminescence assay kit detects glucose levels through a coupled reaction involving glucose dehydrogenase, a reductase, and luciferase. The luciferase reaction exhibits extremely high sensitivity, andpH levelTemperature, experimental system settings, and other parameters all have a significant impact on the reaction; therefore, under most experimental conditions, it is not recommended to directly compare absolute readings of samples. It is advisable to include the same reference compound and solvent control on each assay plate, and to normalize the sample measurements against the reference compound and solvent control before data processing and comparison. Alternatively, glucose standards treated in the same manner as the samples can be included on each plate to quantify the glucose content of the samples.

3.1disturbanceSample processing

UA-Glo®Glucose luminescence assay reagents can be used to analyze a variety of samples, such as extracellular culture media and cell lysates; see the table below for details. The maximum detectable concentration of glucose is50mu calMickeyYou may refer to the table below for appropriate sample processing and dilution, or users can optimize the sample dilution based on their specific experimental conditions.

The inactivation solution used in sample processing is0.6N HCl solution (Cas# 7647-01-0), the neutralization solution is1M Trisalkali(Cas# 77-86-1)Both can be directly prepared at the corresponding concentrations without any adjustments.pH levelThe inactivation solution can rapidly lyse cells and halt cellular metabolism, while also inactivating intracellular enzymes and degrading reducing agents.NAPD(P)H hostedAfter such treatment, the enzymes and reduced forms within the cells…NAPD(P)H hostedThis will not interfere with subsequent glucose measurements. Samples treated with the inactivation and neutralization solution can be tested immediately or stored at-20°CIn future testing.

It is recommended to use tissue homogenate.1Volume of50 mM Tris (pH 7.5),Join1/8The inactivated solution of a certain volume, after homogenizing the tissue, is then added.1/8A neutralizing solution of a certain volume. Or use according to the following table.50 mM Tris (pH 7.5)Immediately after homogenization, add the inactivation solution and then the neutralization solution in sequence. For tissues that are difficult to lyse, an appropriate detergent may be added, for example, at a final concentration of…0.1-0.25%ofDTAB (Dodecyl trimethylammonium bromide)During testing, the tissue homogenate needs to be diluted, which can be done as follows:1Volume of homogenate:1/8Volume of inactivation solution:1/8Prepare the tissue homogenate diluent by mixing the neutralizing solution with the specified volume ratio to dilute the tissue homogenate.

Serum and plasma are required.PBSDilute150x-500xTest again later. If the dilution factor…<10x, an inactivating solution should be added to inactivate endogenous enzymes in serum and plasma, which can be done as follows:1Volume sample:1/8Volume of inactivation solution:1/8Volume neutralization solution ratio dilution factor<10xSerum and plasma samples.

Sample	Sample glucose concentration	Sample processing
Extracellular culture medium	50 μM to 7 mM	PBSDilute100x-250x
Cell lysate	Glucose is rapidly converted into glucose within the cell.-6-Phosphoric acid; therefore, operations must be performed quickly to rapidly terminate cellular metabolism.	1.Discard the culture medium,PBSWash away the remaining culture medium, then add.1Volume(1V)ofPBS 2.Join0.5VThe inactivation solution, shake to mix thoroughly,5 minutes 3.Join0.5VThe neutralization solution, shake to mix well,1 minute
Cells and culture medium	Highest50 μM	1.1VveltCells and culture medium(EnglishOrPBS) 2.Join1/8 VThe inactivation solution, shake to mix thoroughly,5 minutes 3.Join1/8 V-shapedThe neutralization solution, shake to mix well,1 minute
Organization	3mgLiver tissue in1mLshortThe glucose concentration of the homogenate is approximately75 μM	1.1VveltHomogenized tissue 2.Join1/8 V-shapedThe inactivation solution, shake to mix thoroughly,5 minutes 3.Join1/8 VThe neutralization solution, shake to mix well,1 minute
Serum and plasma	3 mM–7 mM	PBSDilute500x

3.2opportunitiesGlucose detection experiment

3.2.1cannot be translatedSample glucose test

1)White(EnglishBottom opaque)closeAdd the sample with an appropriate dilution to the test plate.50mu calLlifEnglish:Per well. The negative control consists of glucose-free culture medium or buffer treated in the same manner as the samples, while the positive control can be a glucose standard treated in the same way as the samples.The luciferase reaction is sensitive to temperature changes. Both the assay reagents and samples should be equilibrated to room temperature, and the temperature should be kept constant throughout the assay. (±1℃)close

2)Take outUA-Glo®After the luciferin detection solution in the glucose luminescence assay kit has completely melted, invert it thoroughly to mix. Take the volume of luciferin detection solution required for the experiment and equilibrate it to room temperature. (22suitable℃-25℃)close.The remaining fluorescein detection solution should be aliquoted as needed and stored at-80℃The fluorescein detection solution should not be subjected to more than [number] freeze-thaw cycles.3EACNext

3)Glucose assay reagent preparation: Before testing, remove the reducing enzyme substrate and place it on ice to thaw. After it has completely melted, proceed as follows:200xregretAdd the luciferin detection solution, which is the glucose assay reagent. The glucose assay reagent should be used immediately after preparation; otherwise, the background signal may increase. Prepare the glucose assay reagent fresh as needed and do not store it.The remaining reductase substrate should be aliquoted as needed and stored at.-80℃

4)Add glucose detection reagent to each well of the experimental plate.50mu calLlivingEnglish:. Sample andGlucose test reagentVolumeRatio is1:1 panicFor example, using a low volume.384-During orifice plate inspection, it can be added.10mu calLlifEnglish:Sample and10mu kaiLlifEnglish:Detect using a glucose test reagent.

5)Medium-speed vibrating plate1 minute, incubate in the dark30 min - 60 min,

6)Read the luminescence signal on a luminometer or multifunctional reader.,It is recommended to add the glucose test reagent after…2-3 hoursInternal reading board

3.2.2Extracellular glucose detection in cell culture medium

1)In96Seed the experimental cells at an appropriate density in a 96-well cell culture plate, for example.5,000-50,000Cells per well, including a medium-only control. The glucose concentration in the cell culture medium should ideally not exceed.7mMairro

2)Perform cell treatment according to experimental requirements.

3)Collect culture medium samples at the corresponding time points as required by the experiment.2-5mu calLlifEnglish:and dilute to98-95mu calL PBSviewIn the middle. DilutedThe sample can be tested immediately or stored.-20°CConduct testing in the future.

4)Further use before testing.PBS 2x-4xDilute the sample to a final dilution of100xReplayOr optimize the dilution factor based on specific experiments.

5)White (EnglishBottom opaque)closeAdd a sample of appropriate dilution to the test plate.50 μLPer well

6)Add to each well.50mu calLlifEnglish:Glucose test reagent,Press [3.2.1cannot be translatedSampleGlucoseDetectionPerform the steps for inspection.

3.2.3Glucose detection in cell lysates

1)In96Seed the experimental cells at an appropriate density in a 96-well cell culture plate, for example.5,000-50,000Cells per well

2)Perform cell treatment according to experimental requirements.

3)Discard the culture medium, and use200 vehiclesmu calLlifEnglish:ColdPBSWash the cells twice to remove residual glucose from the culture medium.

4)Add to each well.25mu calL PBSview

5)Add to each well.12.5mu calLlifEnglish:The inactivation solution, shake to mix thoroughly,5 minutes.PBSAnd the inactivation solution can also be done as2peace:1After mixing, add them together to the experimental plate, i.e., add to each well.37.5mu calLlifEnglish:This mixture, shake the plate.5 minutes

6)Join12.5mu calLlifEnglish:Neutralizing solution,Oscillate and mix.1 minute

7)Add to each well.50mu calLlifEnglish:Glucose test reagent,Press [3.2.1cannot be translatedSampleGlucoseDetectionProceed with the steps for inspection.

3.2.4Homogenization of cells and culture medium glucoseDetection

1)On the day of the experiment, resuspend the experimental cells in glucose-free medium.96Add to each well of the cell culture plate.25 μLCells at an appropriate density. Cells can also be seeded in complete culture medium.96After overnight incubation in a 96-well cell culture plate, discard the culture medium.200 vehiclesmu calLlifEnglish: PBSAfter washing the cells to remove residual glucose from the culture medium, add.25mu calLlifEnglish:Glucose-free culture medium

2)Perform cell treatment according to experimental requirements, such as adding.5mu calLlifEnglish:The test compound, incubation5-15 minutesThe compound needs to be diluted in a glucose-free medium.

3)Add glucose to the glucose-free medium, with a glucose concentration of4xreplayFinal experimental concentration, for example40mM. Add10mu calLlifEnglish:Culture medium containing glucose, based on the above steps.2peaceThe experimental plate has a final glucose concentration of10mM

4)Incubate for a specified time according to experimental requirements. Steps3EACFinal glucose concentration and steps4The incubation time needs to be optimized based on the specific experiment to ensure that the measured glucose concentration is within the desired range.0.05μMemics-50μMemicsBetween

5)Add to each well.5mu calLlifEnglish:The inactivation solution, shake to mix thoroughly,5 minutes

6)Add to each well.5mu calLlifEnglish:Neutralizing solution,Oscillate and mix.1 minute

7)Add to each well.50mu calLlivingEnglish:Glucose test reagent,Press [3.2.1cannot be translatedSampleGlucose[Detection]Perform the steps for inspection.

It is not recommended to mix different batches.
It is not recommended to alter the volume of the test reagent unless it has been rigorously validated.
For research use only