Amplite® Fluorimetric Sphingomyelin Assay Kit *Red Fluorescence*

Protocol summary

1. Prepare SMase working solution (50 µL)
2. Add sphingomyelin standards or test samples (50 µL)
3. Incubate at 37 °C for 1 - 2 hours
4. Add sphingomyelin assay mixture (50 µL)
5. Incubate at RT for 0.5 - 2 hours
6. Monitor fluorescence intensity at Ex/Em = 540/590 nm (cut off at 570 nm)

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

1. SMase stock solution (100X):
Add 50 µL of PBS with 0.1% BSA into the vial of Sphingomyelinase (Component B) to make SMase stock solution (100X).

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. Sphingomyelinase (SMase) working solution:
Prepare SMase working solution by adding the whole content (50 µL) of 100X SMase stock solution into 5 mL of SMase Reaction Buffer (Component D) and mix well. Note: The SMase working solution should be used promptly.

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

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

Table 1. Layout of sphingomyelin standards and test samples in a solid black 96-well microplate. SM = Sphingomyelin Standards (SM1 - SM7, 0.1 to 100 µM), BL = Blank Control, TS = Test Samples.

Table 2. Reagent composition for each well.

1. Prepare sphingomyelin standards (SM), 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 SMase working solution to each well of sphingomyelin 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 SMase 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 sphingomyelin assay mixture to each well of sphingomyelin standard, blank control, and test samples to make the total sphingomyelin assay volume of 150 µL/well. For a 384-well plate, add 25 µL of sphingomyelin assay mixture 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 (cut off at 570 nm).