Evidence for a fence that impedes the diffusion of phosphatidylinositol 4,5-bisphosphate out of the forming phagosomes of macrophages.

Materials Bodipy-TMR-PIP 2 was purchased from Echelon Bioscience (Salt Lake City, UT). The chemical structure of this lipid is illustrated in Gambhir et al. (2004) . TopFluor-PI(4,5)P 2 was purchased from Avanti Polar Lipids (Alabaster, AL). DiIC 12 was purchased from Invitrogen (Carlsbad, CA). Arachidoyl Lyso-PC was purchased from Avanti Polar Lipids. Rhodamine B and human IgG were from Sigma-Aldrich (St. Louis, MO). The 8-μm latex beads were from Bangs Labs (Fishers, IN). Glass capillaries, thin walled with filament inner diameter 1.0 mm, were purchased from World Precision Instruments (Sarasota, FL). Needles were pulled on a Flaming Brown micropipette puller, model P.80/PC, Sutter Instrument (Novato, CA). The 35-mm glass-bottom dishes were purchased from MatTek Corporation (Ashland, MA).

Cell culture

J774A.1 macrophages were purchased from the American Type Culture Collection (ATCC; Manassas, VA). Cells were maintained at 37°C with 5% CO 2 in DMEM supplemented with 10% fetal bovine serum (FBS), 50 U/ml penicillin, 50 μg/ml streptomycin sulfate, and sodium pyruvate. J774A.1 cells used in the examination of PIP 2 and PIP 3 levels with GFP-tagged probes were maintained at 37°C with 5% CO 2 in DMEM with 1.5 g/l sodium bicarbonate from Wisent (St. Bruno, QC, Canada) supplemented with 10% FBS. RAW 264.7 cells used for the actin localization and frustrated phagocytosis experiments were from ATCC and were maintained at 37°C with 5% CO 2 in RPMI-1640 from Wisent supplemented with 5% FBS. RAW 264.7 cells were also used for the PM-GFP FCS and FRAP experiments. They were cultured at 37°C in DMEM (Life Technologies, Invitrogen, Carlsbad, CA) supplemented with 10% FBS (Life Technologies), 50 U/ml penicillin, 50 μg/ml streptomycin sulfate, l -glutamine, and 110 mg/ml sodium pyruvate. As recommended by ATCC, CO 2 was maintained at 10% due to the high sodium bicarbonate level of the Life Technologies media used. Sample preparation Bodipy-TMR-PIP 2 and TopFluor-PIP 2 were injected into J774a.1 cells in the form of arachidoyl Lyso-PC/fluorescent-PIP 2 micelles as described previously ( Golebiewska et al. , 2008 ). The composition of micelles was 82% arachidoyl Lyso-PC and 18% fluorescent-PIP 2 . Lipids were suspended in 1 mM EDTA solutions, warmed to 37–42°C, mixed, and diluted with 1 mM EDTA to a concentration of ∼500 μM, and then sonicated in a bath sonicator for 3–5 s to form the mixed micelles. We used mixed micelles rather than PIP 2 micelles because PIP 2 micelles aggregate in the presence of the 1 mM Ca 2+ present in the bathing solution, causing the pipette tips to clog. DiIC 12 was dissolved in ethanol and added to cells by mock microinjections (with the Pi and Pc set to 0). The final concentration of ethanol in the cell was