May gr nwald s stain solution

Briefly, cells (BxPC-3, 4×10⁵ cells/well; KP-4, 4×10⁵ cells/well; PANC-1, 3×10⁵ cells/well) were seeded and cultured for 24 h in 60 mm-dishes, followed by treatment with lapatinib (10 µM) and FTY720 (10 or 15 µM) at 37°C for 24 h. To detect adherent and detached cells, floating cells in culture medium and trypsin-treated adherent cells were collected. Then, cells were spread onto glass slides using a Cytospin 4 centrifuge (Thermo Fisher Scientific, Inc.) and air-dried. These cells were stained with May-Grünwald's stain solution (Muto Pure Chemicals Co., Ltd.) for 3 min at room temperature. The glass slides were allowed to stand for another 3 min after adding the same phosphate-buffered saline (PBS) volume. After removing the May-Grünwald's stain solution, the cells were stained with diluted (1:20 with water) Giemsa's stain solution (Muto Pure Chemicals Co., Ltd.) for 20 min at room temperature. After washing and drying, the cells were observed using a BZ-X810 digital light microscope (Keyence Corporation) featuring PlanApo 60× NA1.40 (Nikon Corporation).