Amplite® Fluorimetric Sphingomyelinase Assay Kit *Red Fluorescence*

Protocol summary

1. Prepare Sphingomyelin working solution (50 µL)
2. Add SMase standards and/or SMase test samples (50 µL)
3. Incubate at 37°C for 1 - 2 hours
4. Add Sphingomyelinase working solution (50 µL)
5. Incubate at RT for 1 - 2 hours
6. Monitor fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 570 nm)

Important notes
Thaw one vial (or bottle) of each kit component at room temperature before starting your experiment.

1. Sphingomyelinase standard solution (10 U/mL):
Add 20 µL of PBS with 0.1% BSA into the vial of Sphingomyelinase Standard (Component F) to make a 10 units/mL Sphingomyelinase standard solution.

2. Amplite™ Red stock solution (200X):
Add 80 µL of DMSO (Component G) into the vial of Amplite™ Red (Component C) to make 200X Amplite™ Red stock solution. Keep from light. Note: The Amplite™ Red is unstable in the presence of thiols (such as DTT and 2-mercaptoethanol). The final concentration of DTT or 2-mercaptoethanol in the reaction should be lower than 10 µM. Amplite™ Red is also unstable at high pH (>8.5). The reactions should be performed at pH 7 - 8. pH 7.4 is recommended for the assay buffer.

1. Sphingomyelin working solution:
Add 50 µL of Sphingomyelin (Component B) into 5 mL of SMase Reaction Buffer (Component D) and mix well to make Sphingomyelin working solution. Note: The Sphingomyelin working solution should be used promptly.

2. Sphingomyelinase working solution:
Add 5 mL of Assay Buffer (Component E) into the bottle of Enzyme Mix (Component A) and mix them well. Then, add 25 µL of 200X Amplite™ Red stock solution into the bottle of Enzyme Mix solution to make Sphingomyelinase working solution before starting the assay. Note: The Sphingomyelinase working solution should be used promptly and kept from light; longer storage is likely to cause high assay background.

Table 1. Layout of Sphingomyelinase standards and test samples in a solid black 96-well microplate. SMase = Sphingomyelinase Standards (SMase1 - SMase7, 0.078 to 5 mU/mL), BL = Blank Control, TS = Test Samples. 

Table 2.  Reagent composition for each well.

1. Prepare Sphingomyelinase standards (SMase), 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: Treat your cells or tissue samples as desired.
   
   
2. Add 50 µL of Sphingomyelin working solution to each well of Sphingomyelinase standard, blank control, and test samples to make the total Sphingomyelin assay volume of 100 µL/well. For a 384-well plate, add 25 µL of Sphingomyelin working solution into each well instead, for a total volume of 50 µL/well.
   
   
3. Incubate the reaction mixture at 37°C for 1 - 2 hours.
   
   
4. Add 50 µL of Sphingomyelinase working solution to each well of Sphingomyelinase standard, blank control, and test samples to make the total Sphingomyelinase assay volume of 150 µL/well. For a 384-well plate, add 25 µL of Sphingomyelinase working solution into each well instead, for a total volume of 75 µL/well.
   
   
5. Incubate the reaction mixture for 1 - 2 hours at room temperature (protected from light).
   
   
6. Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm).