5% BSA, 0.1% saponin in PBS. Cells were subsequently incubated with primary and fluorescently labelled secondary antibody for 45 min. Unbound antibodies were removed by washing with blocking buffer. Coverslips were washed and mounted using Prolong gold (Invitrogen). Imaging was performed on a Zeiss LSM 510 confocal microscope equipped with a Ar/Kr laser for 488 nm and a He–Ne laser for 543 nm, using Alpelisib a Plan-Apochromat 63×/1.40 oil objective. Microscope parameters were set to detect optimal
signals below the saturation limits. Quantitation of overlapping signals in different channels was done with the colocalization tool of ImageJ and expressed as Pearson’s coefficient (Bolte and Cordelieres, 2006). RBL-2H3 cells (2 × 106) were washed in 1 ml DMEM containing 1% FCS (SDMEM) and incubated for 30 min at 37 °C in 0.5 ml SDMEM containing 50 ng ml−1 IgE anti DNP. RBL-2H3 loaded with IgE anti DNP can be activated by multivalent DNP–HSA conjugate. Cells
were washed, and triplicate samples of 150 μl were incubated for 1 h at 37 °C in SDMEM containing 500 ng ml−1 DNP–HSA. Incubations with HSA (spontaneous release), and 0.2% TX-100 (total lysis) were used as negative and positive control, respectively. 50 μl supernatant was harvested and added to 50 μl 2 mM p-nitrophenyl-N-acetyl-β-d-glucosaminide (Sigma) for 1 h at 37 °C in a 96-well plate. β-hexosaminidase activity Y 27632 was determined colorimetrically at 405 nm after adding 150 μl 0.1 M carbonate buffer pH 10. Alternatively, degranulation was induced by 100 nM
phorbol 12-myristate 13-acetate (PMA, Sigma) and 1 μM ionomycin (Calbiochem) with DMSO as negative control and β-hexosaminidase release was assayed as above. FRAP was determined on live cells using a Zeiss LSM510 microscope with live cell imaging chamber at 37 °C and 5% CO2. RBL-2H3 grown on 25 mm coverslips were transferred to imaging chambers filled with 750 μl SDMEM for FRAP experiments. Activation of cells was done by adding 250 μl SDMEM, 4 μM ionomycin, 400 nM PMA, 800 nM FM4-64 to the live cell imaging chamber. Cells were activated pharmacologically to induce a faster and more homogeneous response. Imaging was started 90 s after the addition of the drugs, when most cells showed first signs of degranulation. Akt inhibitor Frames were recorded in the red (560 nm long pass) and green (505–530 nm band pass) channel every 3 s. Bleach settings were 8 pulses of the 488 laserline at max laser power after the first 5 frames. Cells were imaged for 3 min after bleaching. Recovery was determined as the recovery of the fluorescence in the region of interest corrected for the background signals outside the cells and for loss of fluorescence using a ROI of the whole cell. The signal of the dye FM4-64 was used to distinguish activated from resting cells.