Amplite® Fluorimetric Glucose Quantitation Kit

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.

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.

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

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.

Table 3. Reagent composition for each well

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).