Diff quik solution kit

The upper chamber of a Transwell plate (8-mm pore size; Millipore, Billerica, MA) was coated with 100 μL of Matrigel (Sigma-Aldrich) at 37 °C for 15 min and then incubated at room temperature for 10 min. MDA-MB-231 cells (1.5 × 10⁵ cells/well) were seeded in the Matrigel-coated upper chamber for the Matrigel invasion assay and a non-coated upper chamber filled with serum-free medium for the migration assay. Cells were treated with luteolin for 1 h and then with TPA (50 nM) for 24 h. After incubation for 24 h, the serum-free medium was removed, and the cells on the upper side of the chamber were removed using cotton swabs. The cells that had migrated through the membrane were fixed and stained with a Diff-Quik Solution Kit (#38721; Sysmex, Tokyo, Japan). Images of the membrane were captured using a light microscope. Finally, 50 μL of 10 % acetic acid was added to each well, and the cells were incubated for approximately 1 min. The absorbance at 630 nm was measured using the Apollo LB 9110 microplate reader.

For migration assays, cells (6 × 10⁶ cells/ml) were seeded onto the upper chambers (Corning Inc., USA) in DMEM without serum and treated with ML for 2 h. To the lower chamber, 750 μl of TPA-treated serum-free DMEM (50 nM) was then added. After incubation for 24 h, non-migrated cells were removed from the chamber. Migrated cells were stained with a Diff-Quik Solution kit (Sysmex, Japan). The number of migrated cells was assessed using a microscope. Cells were then dissolved in 10% acetic acid (100 μl) and colorimetrically measured at 620 nm.

To assess colony formation, cells were seeded in a 60-mm dish at a density of approximately 500 cells/dish and treated for 7 days with hesperidin (50 µM) or with SP600125 (5 µM) and PM_2.5 (50 μg/mL) without medium change. The medium was discarded, and the resultant colonies were stained using the Diff-Quik solution kit (Sysmex, Kobe, Japan), according to the manufacturer’s instructions. Colonies containing 50 or more cells were considered viable. Colonies were imaged and quantified using ImageJ (version 1.47; National Institutes of Health, Bethesda, MD, USA).