Isolation of lipid rafts from monocytes. Triton X-100 lysates from monocytes were fractionated by centrifugation over a discontinuous sucrose gradient. Eleven equal fractions were obtained and assessed by Western blotting for the presence of (A) PSGL-1, (B) TF, and (C) CD45. Lipid rafts were identified by the presence of the raft-marker, flotillin-1. (D) Effect of membrane cholesterol depletion with MβCD on the localization of PSGL-1 and TF to lipid rafts. (E) As assessed by flow cytometry, cholesterol depletion did not affect the surface expression of PSGL-1 (top panels) or TF (bottom panels) on LPS-treated THP-1 cells. Background fluorescence was set with a fluorescent mouse IgG control (empty histograms). (F) Effect of membrane cholesterol depletion with 5 mM MβCD on the generation of microvesicles induced by calcium ionophore in THP-1 cells. The results shown are representative of 3 separate experiments. Values are shown as mean plus or minus SD. *P = .04 (n = 4).