Here is a sample protocol for preparation of cell samples to use with our Amplite® Colorimetric Sphingomyelinase Assay Kit (Cat# 13620, 13621):

1. Plate 2~5 x10^5 cells/well (the cell number will need to be optimized for different cell lines).
2. Remove medium, replace with 50 µL PBS.
3. Add 50 µL sphingomyelin working solution; incubate the reaction mixture at 37 °C for 1 - 2 hours.
4. Add 50 µL of sphingomyelinase working solution; incubate the reaction mixture for 1 - 2 hours at room temperature.
5. Monitor the absorbance increase with an absorbance microplate reader at 655 nm.