Effect of ASMase inhibitor treatment on LPS- and LPS+ATP-induced ASMase and TF expression in vivo in monocytes. Mice were treated with saline (Control) or ASMase inhibitor 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 whole blood of these groups of mice were fixed intact in 4% paraformaldehyde, coated on a glass coverslip, and stained for ASMase or TF using rabbit anti-human ASMase that recognizes murine ASMase and rat anti-murine TF mAb (1H1), respectively, followed by fluorophore-conjugated secondary antibodies. Representative images of immunofluorescence staining of TF and ASMase on PBMCs isolated from control mice (A) and mice treated with desipramine (B) or imipramine (C). (D) Colocalization of ASMase and TF in monocytes isolated from control, desipramine-treated (Des), and imipramine-treated (Imi) mice. Cells shown in the images are monocytes. Other PBMCs, whose cell size was much smaller than monocytes and stained very faintly, were not captured in the image. Quantification of fluorescence intensity of immunostaining of TF (E) or ASMase (F) of PBMCs isolated from control mice and mice treated with desipramine or imipramine. (G) Levels of S-ASMase in plasma of control mice and mice treated with desipramine or imipramine. Data shown are mean ± SEM (n = 6 mice). **P <.01, ***P < .001. DAPI, 4′,6-diamidino-2-phenylindole; ns, not statistically significant.