Diff quik solution

To examine CCA cell migration, 48-well micro chemotaxis chambers (Neuro Probe, Gaithersburg, MD, USA) were used. Culture medium containing 10% FBS (50 μl) was added to bottom chambers, which were then covered with collagen coated membranes (Neuro Probe, Gaithersburg, MD, USA). For collagen coating, 5 ml of PBS containing 0.02 μg/μl of collagen was treated for 30 min. Cells (5x10⁴) in 50 μl of medium containing 0.05% FBS were seeded in upper chambers. After 5 to 10 hours, cells that migrated through membrane were prepared by fixing and then staining membranes using Diff-quik solution (Sysmex, Kobe, Japan). The stained membranes were washed and were attached on micro slide glasses (Matsunami glass, Osaka, Japan). Experiments were performed in triplicate. Pictures were taken using a light microscope at 40X and 200X. Total numbers of migrated cells were counted using Adobe Photoshop CS6 software. SCR or Mock values were used as controls to calculate relative cell migration.

Alfaxalone (80 mg/kg, Jurox Pty Ltd., Rutherford, NSW, Australia) was used to anesthetize the mice, and tracheostomy was performed to collect BALF. Briefly, cold PBS (0.7 mL) was delivered into the lungs via injection into the trachea. The cells were transferred onto a slide using Cytospin (1500 rpm, 10 min; Hanil, Republic of Korea) following extraction of BALF from the animals. Each glass slide was stained with Diff-Quik solution (Sysmex, Horgen, Switzerland) to count the inflammatory cells.

To obtain BALF, the tracheas of anesthetized mice were exposed and cut immediately below the larynx. A polyurethane flexible tube (BD Biosciences, San Jose, California, USA) attached to a blunt 24-gauge needle was placed in the trachea, and the lung was lavaged once with 800 μL of sterile phosphate-buffered saline. BALF samples were centrifuged for 5 min at 3,000 rpm and 4 °C. The supernatants were decanted and immediately frozen at −70 °C. The cell pellets were resuspended and washed twice with phosphate-buffered saline. The total cell numbers were counted using a hemocytometer. To determine the differential cell counts, BALF cells were prepared using a cytospin apparatus and stained with Diff-Quik solution (Sysmex Co., Kobe, Japan) in accordance with conventional morphological criteria. At least 200 cells per slide were evaluated to determine the differential leukocyte counts.

The invasive abilities of cancer cells were assessed using 8-μm porous BioCoat Matrigel chamber inserts (BD Bioscience, San Jose, CA, USA). One day after transfection with SCR or DPY30 siRNA, cells were trypsinized, and suspended at a density of 5 × 10⁴ cells/ml in 500 μl of RPMI supplemented with 0.5% FBS and mitomycin C (0.01 μg/ml, Sigma-Aldrich). Cells were then added to chamber inserts and placed in wells filled with 0.7 ml of medium supplemented with 10% FBS as chemoattractant. After incubation for 24 or 48 hrs, non-invading cells on top of the membrane were removed by scraping, and invasive cells on the bottom of the membrane were fixed and stained with Diff-quik solution (Sysmex). Cell numbers in 10 randomly chosen fields were counted using a light microscope. Experiments were performed in triplicate and at least 5 fields were counted per experiment.

To conduct wound healing assay, cells were seeded into 12-well plates and then incubated over 90% confluence. The plate was scratched with pipette tips and washed with PBS. Cells were incubated for 24 h with fresh DMEM medium containing either vehicle alone or ODZ10117. Digital images were obtained using the Leica Application Suite (LAS) Microscope Software (Leica Microsystems).
Invasion assay was performed using a Boyden chamber system (Neuro Probe, Gaithersburg, MD, USA). Growth factor reduced Matrigel (354230, BD Matrigel™, BD Biosciences) was diluted with serum free media with ratio of 1:3. Diluted Matrigel was transferred into 24-transwell (BD 24-well insert, 8 μm pore transparent PET filter) and incubated at least for 4 to 5 h for gelling at 37 °C. Cells in 100 μL DMEM containing 1% FBS were seeded in the upper chamber and incubated for 24 h in the presence of either vehicle alone or ODZ10117. The lower chamber was filled with 500 μL of 10% DMEM containing fibronectin (5 μg/mL, ECM001, Sigma-Aldrich). Matrigel containing upper chamber was rinsed with PBS, fixed, stained with Diff-Quik solution (Sysmex Corporation, Kobe, Japan), and subsequently rinsed with distilled water. The migrated cells were captured using the LAS Microscope Software (Leica Microsystems).