Amplite® Fluorimetric Alkaline Phosphatase Assay Kit *Blue Fluorescence*

Protocol summary

1. Prepare Alkaline Phosphatase working solution (50 µL)
2. Add Alkaline Phosphatase standards and/or test samples (50 µL)
3. Incubate at RT or 37 °C for 10 - 30 minutes
4. Monitor fluorescence intensity at Ex/Em = 360/450 nm (Cutoff = 435 nm)

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

1. MUP Plus™ stock solution (250X):
Add 100 µL of sterile H₂O into the vial of MUP Plus™ (Component A) to make 250X MUP Plus™ stock solution. The MUP Plus™ stock solution should be used promptly.

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

Add 20 μL of 250X MUP Plus™ stock solution into 5 mL  Assay Buffer (Component B) to make Alkaline Phosphatase working solution. Note: Keep from light. Prepare fresh Alkaline Phosphatase working solution for each experiment.

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

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 - 100 mU/mL); BL=Blank Control; TS=Test Samples.

Table 2. Reagent composition for each well.

Run alkaline phosphate assay in supernatants:

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.
   
   
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.
   
   
4. Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 360 ± 10 nm, Emission = 450 ± 10 nm (Cutoff = 435 nm).

Run alkaline phosphatase assay in cells:

1. Treat the cells as desired.
   
   
2. Add equal volume of Alkaline Phosphatase working solution into each cell well (such as 100 µL/96-well plate, or 50 µL/384-well plate). Note: Alternatively, remove the growth medium from the cell plate, and make 1:1 dilution of the 5 mL Alkaline Phosphatase working solution with 5 mL distilled H₂O. Then add 100 µL (for a 96-well plate) or 50 uL (for a 384-well plate) of 1:1 diluted working solution into the cell wells.
   
   
3. Incubate the reaction at the desired temperature for 30 to 60 minutes, protected from light.
   
   
4. Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 360 ± 10 nm, Emission = 450 ± 10 nm (Cutoff = 435 nm).