Confocal laser-scanning microscopy

For imaging fixed cells, THP-1 cells or iBMDMs were seeded at a density of 2 × 10⁵/ml. Eight to 12 hours after transfection, HEK293T cells were washed once with 1× BRB80 buffer, fixed, and analyzed by immunofluorescence microscopy. After activation, THP-1 and iBMDMs were washed once with 1× BRB buffer, fixed, and labeled with antibodies, as described in the immunolabeling and antibodies section. Confocal sections were obtained using an Olympus FV-1000 laser-scanning confocal microscope (Olympus America, Waltham, MA) with Olympus Fluoview version 3.0 software, using 405-nm excitation/425- to 475-nm emission, 488-nm excitation/500- to 545-nm emission, 559-nm excitation/575- to 620-nm emission, and 635-nm excitation/655- to 755-nm emission laser lines.

Images (512 × 512 or 1024 × 1024) were collected at 4 μs/pixel using a 20× [0.75 numerical aperture (NA), air objective], 40× (0.95 NA, air objective), 40× (1.15 NA, water-immersion objective), or 60× (1.2 NA, water-immersion objective) lenses. The images were identically acquired and processed using Adobe Photoshop software. Distributions of fluorescence intensities and colocalization were analyzed by Velocity version 6.2.1 software.