Fig. 2. Plasma membrane PtdIns(4,5)P₂ is transiently depleted during degranulation. (A) Cells were fixed with ice-cold 3% glutaraldehyde and stained either at rest or after stimulation for the indicated times with compound 48/80. First and third row of panels show merged images of 2C11 (red) and Alexa Fluor 647-concanavalin A (blue). Second and fourth row of panels show fluorescence intensity profiles for 2C11. Bar, 10 µm. Ctrl indicates that 2C11 has been omitted during staining. ^*, contaminating neutrophil. (B,C) 2C11 fluorescence was quantified as described in Materials and Methods. (B,C) Mean fluorescence (± s.e.m.) normalised to the resting level (B), distribution of fluorescence at the indicated time points (C); n>100 cells per time point. (D) RPMCs were loaded with unlabelled or [2-³H]inositol, and stimulated for the indicated time period with compound 48/80. Cells were either fixed and stained with 2C11 and quantified as in B (2C11), or extracted and analysed for PtdIns(4,5)P₂ content by HPLC ([2-³H]-PtdIns(4,5)P₂).