Fig. 3. Integrin-activating agonists cause wt LFA-1 to move into lipid rafts. Cells were incubated with no agonist (-), 0.5 mM Mn²⁺ (Mn²⁺), or 100 nM PdBu (PdBu) for 30 minutes at 37°C. (A) Adhesion to ICAM-1. Cells were allowed to bind to plastic-coated ICAM-1 (with or without stimulation) before washing and quantitation of bound cells. (B) Colocalisation of wt LFA-1 with lipid raft patches. After incubation with or without agonists, cells were incubated with TRITC-conjugated Ctx-B, then crosslinked with rabbit anti-Ctx antibody. Cells were fixed and stained with mAbs against LFA-1, followed by Alexa 488-conjugated goat anti-mouse IgG. Data are representative of three experiments. Bar, 10 µm. (C) Colocalisation of LFA-1 with lipid raft patches. Patches of LFA-1 staining were scored into three categories: good, medium, or no colocalisation (Fig. 2). The percentages of cells falling into each category are expressed as averages±s.d. Data are representative of three experiments. Black bars, unstimulated cells; white bars, cells incubated with Mn²⁺; grey bars, cells incubated with PdBu.