Bodipy labeled sphingomyelin

The activity of Asm in colonic tissue was quantified using BODIPY-labeled sphingomyelin as a substrate. After colonic tissue was pulverized and lysed in 250 mM sodium acetate (pH 5) and 1% NP-40, 2 μg protein was incubated with 100 pmol BODIPY-labeled sphingomyelin (Thermo Fisher Scientific, Germany) for 1 h at 37°C in 250 mM sodium actetate (pH 5) and 0.1% NP-40. The reaction was terminated by the addition of chloroform:methanol (2:1, v:v) to extract the lipids. Subsequently, the lower phase containing lipids was collected, dried in a SpeedVac at 37°C, dissolved in 20 μl chloroform:methanol (2:1, v:v) and transferred onto a thin layer chromatography (TLC) plate. Product and uncleaved substrate were separated using chloroform:methanol (80:20, v/v). After separation, spots were imaged using a Typhoon FLA 9500 and quantified with ImageQuant software.

Tissue samples obtained from the reperfused ischemic middle cerebral artery territory and homologous contralateral brain tissue were lysed in 250 mM sodium acetate buffer (pH 5.0) containing 1% NP-40 detergent (Fluka BioChemika, Morristown, NJ, U.S.A.). Cellular membrane integrity was disrupted with a sonicator. After centrifugation for 5 min at 300 g at 4 °C, supernatants were collected. Lysates were adjusted to a specific protein concentration and incubated with 100 pmol BODIPY-labeled sphingomyelin (Thermo Fisher Scientific) in 250 mM sodium acetate (pH 5.0) and 0.1% NP-40 for 1 h at 37 °C. Chloroform:methanol (2:1, v/v) was added, samples were vortexed and centrifuged for 5 min at 15,000 g to achieve a phase separation. The lower phase was collected and concentrated in a vacuum centrifuge (SPC111V, Thermo Fisher Scientific) for 45 min at 37 °C. Lipids were dissolved in 20 µl chloroform:methanol (2:1, v/v) and spotted onto thin layer chromatography (TLC) plates (Macherey Nagel, Düren, Germany). The TLC run was performed with chloroform:methanol (80:20, v/v). TLC plates were analyzed with a Typhoon FLA 9500 scanner (GE Healthcare Life Sciences) and lipid spots were quantified with ImageQuant (GE Healthcare Life Sciences).