Dmi4000 inverted fluorescent microscope

BME26 cells (5 × 10⁵ cells/well) were plated on glass coverslips (Corning^® Costar^®, Cambridge, MA, USA) in 24-well plates, and incubated in complete medium to attach at 34 °C for 24 h. Chemical inhibitors were added at the final concentrations indicated, and 0.05% DMSO was used in negative control wells. After 24 h of treatment, the cells were washed with 0.15 M NaCl, 10 mM sodium phosphate, pH 7.2 (PBS), and immediately fixed on a buffered (PBS) formaldehyde 4% solution for 15 min at room temperature (RT). The cells were then incubated for 20 min (RT) with 200 µL of a solution containing the nuclear marker DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride, molecular Probes, D1306-1 µg/mL), and 1 μL of the F-actin probe phalloidin (Alexa Fluor^® 555, Molecular Probes, A34055-300 units). The cells were visualized using a Leica DMI4000 inverted fluorescent microscope equipped with two A4 (DAPI) filter cubes and N2.1 (Phalloidin). Pictures were obtained with a DFC365 FX camera, and images were mounted into a single file with the help of Adobe Photoshop (CC19.0).

The BME26 cells (5 × 10⁵ cells/well) were plated on glass coverslips (Corning^® Costar^®, Cambridge, MA, USA), then introduced into 24 well plates and incubated in a complete medium, to attach at 34 °C for 24 h. Chemical inhibitors were added at the final concentrations indicated in the cell culture experiments, and 0.05% DMSO was used in the negative control wells. After 24 h of treatment, the cells were washed with 0.15 M NaCl, 10 mM sodium phosphate, and pH 7.2 (PBS), and immediately fixed in a buffered 4% formaldehyde solution (PBS) for 15 min at room temperature (RT). The cells were then incubated for 20 min (RT) in 200 μL of a solution containing the nuclear marker DAPI (4,6-diamidino-2-phenylindole, dihydrochloride, Molecular Probes, D1306-1 μg/mL) and 1 μL of the F-actin probe phalloidin (Alexa Fluor^® 555, Molecular Probes, A34055-300 units). The cells were visualized using a Leica DMI4000 inverted fluorescent microscope equipped with two A4 (DAPI) filter cubes and a N2.1 (Phalloidin) filter.