Enzyme activity of ASM was measured as previously described with minor modifications.38 (link), 39 (link) Briefly, 1 × 106 CD4+ T cells were stimulated with anti-CD3/28 beads (the ratio of bead and cell is 1 : 1; Invitrogen) for the indicated times. For single anti-CD3 or CD28 antibody stimulation, mouse anti-human CD3 or CD28 antibody (10 μg/ml; BD Pharmingen) was mixed with crosslinking antibody (10 μg/ml; BD Pharmingen) at 4 °C for 15 min, and then was introduced into CD4+ T cells. Stimulation was stopped by putting samples on dry ice for 10 s, and the cells were lysed in sodium acetate buffer (50 mM sodium acetate, pH 5.0, 0.5% NP-40).
Ceramide as the lipid cleavage product of ASM was then measured by TLC. Briefly, 10 μg of cell lysates were incubated with sphingomyelin (0.2 mg, dissolved in a mixture of chloroform and methanol (1 : 1)) for 2 h and then loaded onto a TLC plate, followed by chromatographic separation using a solvent system of chloroform and methanol (3 : 2). Ceramide production was then visualized in iodine vapor, identified using a standard run-in parallel, and scanned by a scanner. Relative ceramide levels were evaluated by Image J software (NIH).
Ceramide as the lipid cleavage product of ASM was then measured by TLC. Briefly, 10 μg of cell lysates were incubated with sphingomyelin (0.2 mg, dissolved in a mixture of chloroform and methanol (1 : 1)) for 2 h and then loaded onto a TLC plate, followed by chromatographic separation using a solvent system of chloroform and methanol (3 : 2). Ceramide production was then visualized in iodine vapor, identified using a standard run-in parallel, and scanned by a scanner. Relative ceramide levels were evaluated by Image J software (NIH).
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