A1r fluorescent microscope

Cells plated on gelatin-coated glass slides (Nunc Lab-Tek II Chamber Slide System) were allowed to attach overnight. Cells were fixed for 20 minutes in 4% Paraformaldehyde (PFA) (Electron Microscopy Sciences, Hatfield, PA, USA), then incubated in blocking solution consisting of 2% normal donkey serum, 1% BSA, 0.1% Triton X-100, 0.05% Tween 20, 0.05% sodium azide in 1× PBS (w/o Ca2+/Mg2+), pH 7.2 for 30 minutes, followed by 30-minute blocking in Protein Block, Serum-free (Dako, Carpinteria, CA, USA) + 0.1% Triton X-100. Sheep anti-Rab27a (Thermo Fisher) or an equivalent concentration of sheep IgG (Thermo Fisher) was diluted 1:40 in donkey serum blocking solution for 1 hour at room temperature, followed by Alexa Fluor 488-conjugated donkey anti-sheep IgG (Thermo Fisher). Cells were mounted in ProLong Gold Antifade with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher) and visualized on an inverted Nikon A1R fluorescent microscope. Identical imaging parameters (objective, light intensity, gain, exposure) were used between slides and samples.

Cells were seeded into tissue culture-treated 0.4 mm height flow chambers (Ibidi, Fitchburg, WI, USA) and allowed to reach confluence for 3 days. Cells were stimulated with shear stress (5 dyne/cm²) for 48 hours at 37°C, 5% CO₂ by perfusing complete cell culture media with a peristaltic pump and an inline pulse dampener. Control cells were maintained in static (no flow) conditions. Following stimulation, cells were gently washed in PBS, fixed in 4% PFA, incubated with Acti-stain 488 phalloidin (Cytoskeleton, Inc., Denver, CO, USA). Slides were filled with ProLong Gold Antifade with DAPI (Thermo Fisher) and visualized on an inverted Nikon A1R fluorescent microscope. Binarized nuclear images were analyzed using the ImageJ ellipse function to quantify the orientation of the nuclear long axis relative to the flow direction. A total of 3500 to 5600 nuclei were analyzed per condition.