Bio coattm growth factor reduced matrigeltm invasion chamber

The 2.5 × 10⁴ cells non stimulated (ctrl) and 0.1 mg/mL 5-FU, 0.1 mg/mL 5-FU + 0.3 µg/mL F6 and 0.3 µg/mL F6 as single agent respectively, were placed in 500 μL DMEM + 0.1% FBS medium (DMEM F12 + 0.1% horse serum + 10 ng/mL TGF-β1 in case of MCF-10A cells) in the upper side of 8-µm filters (BD Bio-CoatTM growth factor reduced MATRIGEL^TM invasion chamber, BD Biosciences-Discovery Labware, Two Oak Park, Bedford, MA, USA) (upper chamber) and placed in wells of a 24-well plate (Falcon, BD Biosciences) (lower chamber), containing 0.8 mL of DMEM 10% FBS medium (DMEM F12 + 10% horse serum in case of MCF-10A cells). After 24 h of incubation, the invasive cells on the lower surface of membranes were fixed, stained with Hemacolor^® (HX54775574, Merck, Darmstadt, Germany) and examined microscopically. Cellular invasion was determined by counting the number of cells on membranes in at least 4–5 randomly selected fields using a Zeiss Axiovert 10 optical microscope. For each data point, four independent experiments in duplicate were performed.

Invasion assays were carried out using BD BioCoat^TM Growth Factor Reduced MATRIGEL^TM Invasion Chamber as per manufacturers’ instructions (BD Biosciences, San Jose, CA, United States). To determine the average number of invading cells, inserts were then stained with crystal violet and viewed under the Nikon Eclipse 90i microscope and the number of invaded cells in 9 fields, were counted at 100× magnification. Mean values of triplicate experiments were calculated and results subjected to t-test.