Matrigel invasion chamber

Cell invasion was analyzed using Matrigel invasion chambers (Becton Dickinson, Franklin Lakes, NJ, USA) following the manufacturer's protocol. Briefly, cells were washed twice with PBS and were then resuspended in RPMI at the appropriate density according to the specific experiment. Equal volumes (0.5 mL/chamber) of the cell suspensions were seeded in the Matrigel invasion chambers (Becton Dickinson), which were placed in 24-well culture plates containing 0.75 mL/well of culture medium supplemented with 10% fetal bovine serum. Cells that had invaded to the underside of the inserts after 24-h incubation were stained and quantified by counting the cells from five microscopic fields. For cell migration assay, cells growing as a monolayer on collagen-coated coverslips were scratched manually with a plastic pipette tip. Wound healing was monitored at the indicated time points after scratching. For analysis of cell adhesion, 5 × 10³ cells were seeded on 96-well plates and incubated for 60 min at 37°C. The bound cells in each well were lysed, stained with 0.2% crystal violet, and quantified by spectrophotometry at OD 595 nm.

The invasiveness of the MDA-MB-231 cells was evaluated using a BD Matrigel Invasion Chamber (8-μm pore size; BD Biosciences, Bedford, MA, United States). Briefly, cells were seeded at a density of 1.0 × 10⁵ cells per well, into the upper chamber of the insert, and treated with less than 1% ethanol, as a vehicle control, or the indicated concentrations of OSEE. DMEM supplemented with 10% fetal bovine serum was placed into the bottom wells of the chamber as a chemo-attractant and then incubated at 37°C for 24 h. Cells were then washed, and non-penetrating cells were removed from the upper surface of the filter with a sterile cotton swab. Cells that had penetrated through the Matrigel to the lower surface of the insert were fixed with 4% formaldehyde, stained with DAPI, and counted under a fluorescence microscope. Assay was repeated three times and data were presented as mean values ± SEM.

A density of 50000 cells was seeded into the inserts of a BD Matrigel^™ invasion chamber (BD Biosciences) in serum-free DMEM. Medium containing 10% FBS was placed in the lower chamber as a chemoattractant. After 48 hours, inserts were fixed and stained with crystal violet, non-migrated cells removed using cotton swabs and images captured using an Olympus CKX41 microscope (Olympus, Tokyo, Japan), with a 20X LCAch objective and registered using analysis getIT software (Olympus)