Amplite® Fluorimetric Alkaline Phosphatase Assay Kit *Green Fluorescence*

1. Prepare Alkaline Phosphatase standards or test samples (50 µL)
2. Add Alkaline Phosphatase working solution (50 µL)
3. Incubate at RT or 37°C for 10 - 30 minutes
4. Monitor fluorescence intensity at Ex/Em = 490/525 nm (Cutfoff =515 nm)

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

For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html

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

Add 100 µL of DMSO (Component D) into the vial of FDP (Component A) to make 250X FDF stock solution. The FDP stock solution should be used promptly.

Add 100 µL of distilled H₂O with 0.1% BSA (H₂O - 0.1% BSA) into Alkaline Phosphatase Standard (Component C, 10 units) to generate 100 units/mL Alkaline Phosphatase standard solution.

Note        The Alkaline Phosphatase standard solution is not stable.

Add 20 μL of 250X FDP stock solution into 5 mL of Assay Buffer (Component B) and mix well to prepare Alkaline Phosphatase working solution.

Note        Keep from light.

Table 1. Layout of Alkaline Phosphatase Standards and test samples in a solid black 96-well microplate. AS = Alkaline Phosphatase Standards (AS1 - AS7, 0.1 to 100 mU/mL); BL=Blank Control; TS=Test Samples.

Table 2. . Reagent composition for each well.

1. Prepare Alkaline Phosphatase standards (AS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.

   Note        Prepare the cell or tissue samples as desired. Unused serial dilutions of Alkaline Phosphatase standard should be discarded.

2. Add 50 µL of Alkaline Phosphatase working solution to each well of Alkaline Phosphatase standard, blank control, and test samples to make the total Alkaline Phosphatase assay volume of 100 µL/well. For a 384-well plate, add 25 µL of Alkaline Phosphatase working solution into each well instead, for a total volume of 50 µL/well.
3. Incubate the reaction at the desired temperature for 10 to 30 minutes, protected from light. Optional: Add 50 µL/well (for a 96-well plate) or 25 µL/well (for a 384-well plate) of Stop Solution (Component E) at the end of 30 minutes incubation.
4. Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 490 ± 10, Emission = 525 ± 10 nm (Cutoff =515 nm).

1. Treat the cells as desired.

2. Remove the growth medium completely from the cell plate.

   Note        It is important to remove the growth medium completely from the cell plate due to the interference of the growth medium with the FDP.

3. Make 1:1 dilution of the 5 mL Alkaline Phosphatase working solution with 5 mL distilled H₂O.
4. Add 100 µL (for a 96-well plate) or 50 µL (for a 384-well plate) of 1:1 diluted Alkaline Phosphatase working solution into the cell wells.
5. Incubate the reaction at the desired temperature for 30 to 60 minutes, protected from light. Optional: add 50 µL/well (for a 96-well plate) or 25 µL/well (for a 384-well plate) of Stop Solution (Component E) at the end of 30 minutes incubation.
6. Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 490 ± 10, Emission = 525 ± 10 nm (Cutoff = 515 nm).