24 well transwell chamber

A 24-well Transwell chamber (Greiner Bio-One, Dutscher, France) with a 8-μm pore PET membrane, was used to perform the migration assay and coated with fibronectin at 7 μg/mL. The lower chamber was filled with 600 μL RPMI 1640 conditioned medium (made with NIH3T3 cell line). Then, 200 μL B16F1 or B16F10 melanoma cells suspension (2.5 × 10⁵ cells/mL with 0.5% FBS), containing 5% of homeopathic dilution, were added to the insert. The cells were allowed to migrate at 37 °C with 5% CO₂ over 6 h. The inserts were washed in PBS and fixed with methanol for 15 min. Non-migrating cells were removed from the upper surface of the inserts by gently scrubbing with a cotton-tipped swab. Each PET membrane were cut and stained with mounting medium DAPI (ProLong Gold DAPI, ThermoFischer), between blades and slats. For counting, 10 pictures were taken per membrane, and each condition was made in duplicate. Experiments were performed at least in triplicate.

Cell migration was measured by a 24-well trans-well chamber (8 µm pore size, Greiner Bio-one). After 24 h of MgCl₂ and/or cisplatin treatment, 2 × 10⁴ cells suspended in 100 μl serum-free DMEM were seeded into the upper chamber of each well, and 600 μl medium containing 2.5% FBS was added to the lower chamber. After a 24 h incubation, the chambers were washed thrice with PBS to wash away the cells remaining in the upper membrane while cells that migrated were fixed in methanol for 30 min, stained with 0.1% crystal violet for 15 min at room temperature and viewed under a microscope at 200× magnification.

Transwell invasion assays were performed using a 24-well transwell chamber (Greiner bio-one, Switzerland). First, Matrigel (BD Bioscience, USA) was applied to the upper ventricular surface of the Transwell chamber basement membrane, and then 2 × 10⁵ A498 and 786O cells were suspended in 0.2 ml of serum-free medium and added to the insert. The lower compartment was supplemented with 0.5 ml of medium containing 10% FBS as a chemical attractant. After incubating for 48 h at 37 °C, the cells on the upper surface of the membrane were carefully removed with a cotton swab, and the cells on the lower surface were continuously fixed with 100% methanol, and then stained with 0.5% crystal violet. Three random fields of magnification of 200X were selected for each insert, and the number of cells was counted under an optical microscope (Olympus, Japan).