Phalloidin california red conjugate

MDMs were differentiated on glass coverslips as described above. For actin filament staining, the cells were incubated with 5 µM LPA species for 30 min, fixed with 4% paraformaldehyde for 10 min at room temperature, and permeabilized with 0.3% Triton X100 for 5 min. Actin filaments were stained with Phalloidin-California Red Conjugate (1:1000; AAT Bioquest, Sunnyvale, CA, USA) for 30 min at room temperature. Glass coverslips were mounted on microscope slides using mounting medium with DAPI (VEC-H-1200, Vector, Burlingame, CA, USA) and sealed with nail polish. Images were taken at 40× magnification and processed on a widefield microscope (Leica DM5500, Leica Microsystems, Wetzlar, Germany). Images of three random fields per sample were analyzed with imageJ 1.54 (https://imagej.net/ij/).

MDMs were differentiated on glass coverslips as described above. For actin filament staining the cells were incubated with 50 µM AA for 24 h, fixed with 4 % paraformaldehyde for 10 min at room temperature and permeabilized with 0.3% Triton X100 for 5 min. Actin filament was stained with 1:1000 diluted Phalloidin-California Red Conjugate (AAT Bioquest, Sunnyvale, USA) for 30 min at RT. Glass coverslips were mounted onto the microscope slides using a drop of mounting medium with DAPI (VEC-H-1200, Vector, Burlingame, USA) and sealed with nail polish. Images were taken at 100× magnification and processed on a widefield microscope (Leica DM5500, Leica Microsystems, Wetzlar, Germany).

Macrophages seeded onto titanium discs (7.5 × 10⁴ cells/disc) were incubated for 24 h, after which viability was assessed. The discs were washed in PBS to remove nonadherent cells before incubation with 250µL of alamarBlue™ (Thermo Fisher Scientific, Seventeen Mile Rocks, QLD, Australia) diluted in complete media for 2 h. The fluorescence at 560/590 nm (excitation/emission) was subsequently measured using a spectrophotometer. To visually assess cell morphology following attachment and culture on the titanium surfaces, titanium discs with adherent macrophages were sputter-coated with gold and imaged using a scanning electron microscope (JCM-5000, Jeol, Tokyo, Japan). The diameter of the adherent cells was also quantitated using confocal microscopy imaging (Nikon A1, Tokyo, Japan) and subsequent image J analysis. The macrophages were fixed with 4% paraformaldehyde before staining the actin filaments with Phalloidin-California Red Conjugate (AAT Bioquest, Sunnyvale, CA, USA) and the cell nuclei with DAPI.