Giemsa s stain solution

Cells were seeded onto a 60-mm dish at 1×10⁶ cells /dish and treated with various reagents for 48 h. After treatment with LPZ and/or AZM, the adherent cells were harvested by trypsinization. Cell spreads were prepared on glass slides using a Cytospin 4 centrifuge (Thermo Fisher Scientific, Inc.) [1,000 × g, for 5 min, at room temperature (RT)]. May-Grünwald-Giemsa staining was performed with May-Grünwald's stain solution (without dilution; cat. no. 15053; Muto Pure Chemicals) for 3 min at RT followed with Giemsa's stain solution (1 drop/1 ml H₂O; cat. no. 15003; Muto Pure Chemicals) for 15 min at RT. Glass slides were examined under a digital light microscope (BZ-8100; Keyence Corporation) (objective magnification, ×100). Representative images were selected.

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).