Latex bead phagocytosis assay

641 nm 1.75-µm Fluoresbrite carboxy latex microparticles (17797-1, Polysciences) were untreated or coated overnight at 4 °C with 30 µg/ml LPS in PBS. The beads were washed ten times in large volumes of PBS containing 1% FBS for removal of unbound LPS. BMDMs were cultured on coverslips in six-well dishes and then were put on ice for 10 min. Microparticles were added at a concentration of approximately three to five beads per cell and were allowed to settle on the cells for an additional 10 min on ice. The plates were warmed to 37 °C by adding warm medium for the appropriate time to allow bead phagocytosis followed by cellular fixation with 3.7% paraformaldehyde for 20 min at room temperature. The fixed cells were permeabilized with 0.1% Triton X-100 and then stained with anti-RFP (1:100 dilution; ab62341; Abcam) and anti-TLR4 (1:100 dilution; 19811-1-AP; Proteintech) for 30 min, followed by incubating with the secondary antibodies (Alexa Fluor 488-conjugated anti-mouse IgG (A21202) and Alexa Fluor 555-conjugated anti-rabbit IgG (A31572) (all from Invitrogen)) for another 1 h at 4 °C. Subsequently, the cells were washed with PBS three times and mounted with pure glycerin. All images were collected with a Precision DeltaVision-OMX Super-Resolution Microscope (GE OMX V4).