Biocoat matrigel invasion chamber kit

The in vitro cell invasion assay was carried out using a Corning BioCoat Matrigel Invasion Chamber kit (354480; Corning, Labware, Inc., Two Oak Park, MA, USA). HeLa cells suspended in 0.5 mL serum-free Dulbecco’s modified Eagle’s medium (DMEM) were plated into the top of the invasion inserts. The 0.75 mL of 10% fetal bovine serum-supplemented DMEM was added into the well of the 24-well plate as a chemoattractant. After 48 h of incubation, the inserts were transferred to new plates and washed with PBS. Fixation of the cells suspended in the Matrigel basement membrane matrix at the bottom of each insert was performed using methanol:acetone (1:1). Then, cells were stained overnight with 0.5% Crystal Violet. The excessive stain was removed by rinsing in distilled water. The inserts were air-dried, visualized and photographed under the microscope (Nikon TS100-F, Nikon, Tokyo, Japan). DMSO was used as a vehicle control.

Invasion chamber inserts from BioCoat Matrigel Invasion Chamber kit (Corning, NY) were used. The inserts were thawed and rehydrated in a 24 well plate using serum free media both beneath and inside the insert for 2 hours. The inserts were placed on top of 750μl of serum containing media in a new well of the 24 well plate. 5 × 10⁴ of RJ345, RJ423EV, or RJ423200c cells were each seeded in serum free media on top of the inserts. After 24 hours, cells that had migrated to the opposite sided of the chamber were fixed in 100% methanol for 2 minutes at room temperature and then stained with toluidine blue for 2 minutes at room temperature. Cells were imaged using an inverted Olympus IX71 microscope with Q Imaging software (Q Imaging). The number of cells able to migrate and invade through the insert were counted manually using the ImageJ software (National Institutes of Health). Experiments were performed in triplicate (n=3).