Amplite® Fluorimetric Alkaline Phosphatase Assay Kit *Near Infrared 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 30 to 120 minutes
4. Monitor fluorescence intensity at Ex/Em = 620/660 nm (Cutoff = 630 nm)

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

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

2. Alkaline Phosphatase standard solution (100 U/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 a 100 units/mL Alkaline Phosphatase standard solution.  Note: The Alkaline Phosphatase standard solution is not stable.

For one 96-well plate, add 20 μL of 250X SunRed™ Substrate stock solution to 5 mL of Assay Buffer (Component B) and mix well to prepare Alkaline Phosphatase working solution . Note: Keep from light and prepare fresh reaction mixture 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, 0.3 to 300 mU/mL); BL=Blank Control; TS=Test Samples.

Table 2. Reagent composition for each well.

Run Alkaline Phosphatase 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 for 30 to 120 minutes at the desired temperature, protected from light.
   
   
4. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 620/660 nm (Cutoff = 630 nm).

Run Alkaline Phosphatase assay in cells:

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 SunRed™ Substrate.
   
   
3. Make 1:1 dilution of the 5 mL Alkaline Phosphatase working solution with 5 mL distilled H₂O.
   
   
4. Add 100 µL (96-well plate) or 50 uL (384-well plate) of 1:1 diluted Alkaline Phosphatase working solution into each cell well.
   
   
5. Incubate the reaction for 30 to 60 minutes at the desired temperature, protected from light.
   
   
6. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 620/660 nm (Cutoff = 630 nm).