Total macrophage proteins were extracted using the Cell Lytic™ Cell Lysis Reagent (Sigma, St. Louis, MO, USA). ASM activity assay was performed according to a previously described procedure21 (link)). Briefly, 3 µl cell lysis was mixed with 3 µl ASM buffer [200 µm BODIPY®-labeled C12-Sphingomyelin (Thermofisher Scientific, Waltham, MA, USA)]. Then, it was diluted in an assay buffer [0.2 M sodium acetate (pH=5.0) containing 0.2 mM ZnCl2 and 0.2% Igepal CA-630]. Finally, it was incubated at 37 °C for 20 h. Furthermore, the reaction was terminated by adding ethanol. The hydrolytic product was detected and quantified using ultra performance liquid chromatography (UPLC) system (Acquity UPLC H-class, Waters, Milford, MA, USA). In this system, we used a reversed phase column (Acquity BEH Amide, 2.1 × 50 mm, 1.7 µm, Waters, USA).
Bodipy labeled c12 sphingomyelin
Manufactured by Thermo Fisher Scientific
Sourced in United States
BODIPY®-labeled C12-Sphingomyelin is a fluorescent lipid analogue. It is composed of a C12 fatty acid chain and a BODIPY fluorescent label.
Automatically generated - may contain errors
2 protocols using bodipy labeled c12 sphingomyelin
1
Quantification of Acid Sphingomyelinase Activity
2
Quantitative UPLC-based aSMase Assay
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An ultra performance liquid chromatography (UPLC) system was used to detect aSMase activity [15 (link)]. Briefly, 3 µl of plasma or tissue lysis was mixed with 3 µl of aSMase buffer [200 µm BODIPY-labeled C12-Sphingomyelin (Thermofisher Scientific, MA, U.S.A.)]. Then, we transferred this mixture into the assay buffer [0.2 M sodium acetate (pH = 5.0) containing 0.2 mM ZnCl2 and 0.2% Igepal CA-630] and mixed it well. Subsequently, the samples were incubated at 37°C. After 20 h, the reaction was stopped by adding 100 µl of ethanol. Finally, the product was detected and quantified using the UPLC system (Waters, MA, U.S.A.).
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