Dulbecco phosphate buffered saline (dpbs)

To evaluate the P-selectin ligand expression on AsPC-1, Capan-2, and MIA PaCa-2 cells, respectively, 0.3 µg recombinant human P-selectin Fc Chimera Protein in 50 µL DPBS (R&D systems) was immobilized in each well of a Nunc MaxiSorp flat-bottom 96-well plate for 12 h at 4 °C. Afterwards, unspecific binding sites were blocked with 250 µL BSA solution (4% in DPBS) per well for 4 h and wells were rinsed with 250 µL DPBS thrice. Tumor cells were detached with EDTA (0.2 g/L EDTA × 4 Na, Sigma Aldrich) for 10 min at 37 °C, labeled with Calcein-AM (1 µM Calcein-AM concentration) for 1 h, and rinsed twice with warm DPBS buffer. Then, 5 × 10⁵ tumor cells in 100 µL DPBS were added in each well and the plate was gently shaken on a plate shaker for 2 h. Unbound cells were removed with DPBS and adherent cells were lysed with 1% Triton X-100 (v/v) in DPBS (Merck). Calcein-AM was quantified in a FLUOstar Optima plate reader (BMG Labtech, Ortenberg, Germany) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm.

Cells plated on chamber slides were rinsed by DPBS (Thermo Fisher Scientific, Waltham, MA, USA), then fixed with 4% paraformaldehyde for 15 min, followed by permeabilization with PBST (DPBS containing 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA)) for 15 min. After washing with DPBS, cells were blocked in 5% BSA (R&D Systems, MN, USA) for 30 min, then incubated with the diluted primary antibodies (BD Biosciences, Franklin Lakes, NJ, USA) in 5% BSA at 4 °C overnight. After a thorough washing, the cells were blocked with 5% BSA for 30 min again and then incubated with the secondary antibodies for 1 h at room temperature in the dark. Nuclei were stained with DAPI (Sigma-Aldrich, St. Louis, MO, USA) for 5 min. Confocal (Leica, Wetzlar, Germany) photography was used to analyze the stained cells.