Phagosome and sucrosome analyses

For phagocytosis assays, RAW264.7 cells were plated on 18-mm glass coverslips at a concentration of 2 × 10⁵ cells · ml⁻¹ and grown for 16–24 h. Cells were transfected as described above with the constructs indicated in the text. The day of experiments, SRBCs were prepared for use in phagocytosis. Briefly, 100 µl of SRBC suspension was washed with 3× with PBS and labeled with Alexa Fluor 405 NHS ester for 20 min, shaking at room temperature. SRBC were then opsonized with 2 µl of rabbit anti-SRBC IgG at 37°C for 1 h. Prepared SRBCs were washed 3× with PBS and resuspended to a final volume of 1 ml in PBS. After 1:10 dilution in PBS, 25 µl of this suspension was added to the RAW264.7 cells. Alternatively, FITC-labeled zymosan was diluted to 10 mg · ml⁻¹ in PBS and opsonized by incubation with human IgG (final IgG concentration, 5 mg · ml⁻¹) for 30 min at room temperature. Prepared zymosan was then washed 3× with PBS and resuspended to a final volume of 20 µl in PBS. 1 µl of this suspension was added to RAW264.7 seeded onto coverslips. In all cases, phagocytosis was synchronized by sedimenting particles onto the cells using centrifugation at 300 × g for 1 min. After phagocytosis, monolayers were fixed in 3% PFA for 10 min at room temperature and stored in PBS until used. FITC-zymosan phagocytosis, which was used to determine the rate of acidification of the nascent phagosome, was imaged immediately after sedimentation of the particles.

To generate sucrosomes, transfected RAW264.7 cells were incubated for 16–24 h in RPMI-1640 medium containing L-glutamine, 10% heat-inactivated FCS, and 30 mM sucrose at 37°C under 5% CO₂. The next day, cells were washed 3× with PBS, placed in RPMI-1640 medium containing L-glutamine and 10% heat-inactivated FCS, and used for experiments as described in the text. After experiments, monolayers were fixed in 3% PFA for 10 min at room temperature and stored in PBS.