Amplite® Fluorimetric Glucose and Sucrose Assay Kit

1. Add sucrose standards or test samples (50 µL).

2. Add invertase working solution to each well (10 µL).

3. Incubate at 37°C for 30 minutes.

4. Add glucose working solution (40 µL) to each well.

5. Incubate at 37°C for 30 minutes, protected from light.

6. Monitor fluorescence at Ex/Em = 540/590 nm, cutoff 570 nm.

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

Important Note: Before starting the experiment, thaw all of the kit components at room temperature.

1. Add 50 µL of DMSO (Component F) to the vial of Amplite® Red (Component A) to make a 100X Amplite® Red stock solution.

   Note: The stock solution should be used immediately. Any remaining solution should be made as single-use aliquots and stored at -20 °C. 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.

1. Add 100 µL of Assay Buffer (Component B) to the vial of Invertase (Component C) to make a 200X invertase stock solution.

   Note: Any unused 200X invertase stock solution should be made as single-use aliquots and stored at -20 °C.

1. Add 1 mL of ddH2O to the vial of Sucrose Standard (Component E) to make a 10 mM sucrose standard solution.

1. To make an invertase working solution, add 50 µL of 200X invertase stock solution to 950 µL of Assay Buffer (Component B). Prepare the amount of working solution as needed.

1. Add 5 mL of Assay Buffer (Component B) to the bottle of Glucose Enzyme Mix (Component D), and mix well.

2. Add 50 µL of 100X Amplite® Red stock solution to the bottle and mix well to create the Glucose working solution.

   Note: This working solution is enough for one 96-well plate. It is unstable at room temperature and should be used promptly within 2 hours and avoided exposure to light. Alternatively, one can make a 25X Glucose Enzyme Mixture stock solution by adding 200 μL of H2O to the bottle of Component A and then prepare the working solution by mixing the stock solution with assay buffer (Component B) and 100X Amplite Red stock solution proportionally.

Table 1. The layout of Sucrose standards and test samples in a clear bottom 96-well microplate. STD=Sucrose Standards (STD1-STD7, 0.14 to 100uM), BL=Blank Control, TS=Test Samples.

Table 2. Reagent composition for each well.

1. Prepare sucrose standards (STD), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For each test sample requiring sucrose detection, prepare 2 sets of replicates: one set for sucrose/total glucose detection, and one set for glucose detection.

2. Add 10 µL of invertase working solution to each of the sucrose standards and sucrose/total glucose detection wells. Add 10 µL of Assay Buffer (Component B) to the glucose detection wells.

3. Incubate the plate at 37°C for 30 minutes.

4. Add 40 µL of the glucose working solution to each of the wells. Mix well by using a horizontal shaker or by pipetting.

5. Incubate the plate at 37°C for 30 minutes, protected from light.

6. Monitor the fluorescence intensity with a fluorescence microplate reader at Ex/Em = 540/590 m, cutoff 570 nm.

The standard curve of Sucrose is shown above in Figure 1. To calculate the sucrose concentrations of the samples according to the standard curve, we recommend using the Online Linear Regression Calculator which can be found at:

https://www.aatbio.com/tools/linear-logarithmic-semi-log-regression-online-calculator