Neutrophil Polarized Morphology Analysis by Immunofluorescence

For the analysis of actin polymerization and polarized morphology, neutrophils were seeded at 1-2x10⁶ cells/ml in 1 ml volume in a 35 mm dish with 20 mm #1.5 coverslip (MatTek). The coverslips were pre-coated with 25 µg/ml fibrinogen (F4883, Sigma-Aldrich) in DPBS for 1 hr at 37°C. Neutrophils were incubated 5-10 min in the incubator at 37°C to allow floating cells to settle down on the coverslip. Cells were then stimulated with an equal volume of TCM, media control, positive control fMLF or vehicle control DMSO and were further incubated for 15-20 min at 37°C. Following stimulation, the entire liquid medium was gently discarded from each dish and the adherent cells were immediately fixed with 4% PFA (Electron Microscopy Sciences), 0.1% glutaraldehyde (G5882, Sigma-Aldrich), 5% sucrose (BP220, Fisher Scientific), and 0.1M cacodylate (11652, Electron Microscopy Sciences), for 15 min at room temperature (RT). Once fixed, cells were washed with 0.1M glycine (G8898, Sigma Aldrich) in DPBS to quench the autofluorescence of glutaraldehyde. Washed cells were next permeabilized with 0.05% saponin (84510, Sigma Aldrich) in 3% BSA-containing DPBS for 15 min at RT and stained with phalloidin-TRITC (P1951, Sigma Aldrich) and DAPI (D9542, Sigma Aldrich). Cells were imaged using a 63x objective lens on a Zeiss LSM880 confocal microscope. Cells in three to five different fields across the coverslip were captured randomly per condition in each experiment. F-actin polymerization and cell circularity were measured using the ImageJ image analysis software. Briefly, integrated density, which is a product of cell area and mean fluorescence intensity, and circularity were obtained from ImageJ after manually identifying the boundary of each cell in a given field. Polarization index was calculated by normalizing the integrated density of each cells with respect to the positive control (fMLF) in each experiment.