Figure 3.

LPS treatment reduces SM levels in monocytes: effect of ASMase inhibitors on SM levels in monocytes. Mice were treated with saline (Control) or ASMase inhibitors desipramine or imipramine and then challenged with saline, LPS, or LPS followed by ATP, as described in the legend for Figure 1. PBMCs isolated from mice were stained for SM using lysenin, which specifically binds SM. The cells were incubated with lysenin (0.5 µg/mL) for 60 minutes in phosphate buffer containing 2% bovine serum albumin. The bound lysenin was detected using anti-lysenin antiserum (200× diluted), followed by secondary antibodies conjugated with AF567 fluorophore. (A) Representative images; original magnification ×126. (B) Quantified values for fluorescence intensity associated with monocytes (identified having a larger nucleus using DAPI staining; n = 30-70 cells). ***P < .001.