6 well cell culture dish

To measure cell proliferation in vitro, 1.0 × 10⁴ human PDAC cells were plated in a 6-well cell culture dish (Corning, Cat #07-200-83) and grown in culture conditions listed above. At days 1, 3, 7, 10 and 14, cells were washed with 1× PBS (Gibco, #10010-023) and detached using trypsin-EDTA (Gibco, #25200-056) until cells were free-floating. Then, an equal volume of media with foetal bovine serum (FBS, Atlanta Biologicals, #S11195) was used to create a single-cell suspension. The concentration of cells was determined using a haemocytometer (Reichert, Depew, NY, USA, Cat #Z359629). Both live and dead cells were counted as distinguished by exclusion or passive uptake of trypan blue solution (Sigma, Cat #T8154). Experiments were conducted in triplicate and standard errors were calculated.

Canine skin fibroblasts were generated from skin samples from three different healthy research bred dogs (see above) as described previously (21 (link)). Skin biopsy was taken using a 6 mm skin biopsy punch (Miltex, York, PA). and placed in a 6 well cell culture dish (Corning Inc. Corning, NY) and cultured in complete DMEM. Fibroblasts outgrowths were collected and used for in vitro assays.

BxPC-3 cells transfected with anti-TRPM7 shRNA or non-targeting control shRNA, and non-transfected cells, were cultured in RPMI 1640 medium containing 10% heat-inactivated FBS at 37°C for 24 h. The cells were washed in phosphate-buffered saline (PBS) and incubated in serum-free medium containing 0.1% bovine serum albumin (Sigma®) for another 48 h. The cells were then seeded at 3×10⁴ cells in 1 ml medium in each well of a 6-well cell culture dish (Corning). Trans-well inserts with 8 µm filters (Greiner Bio-One, VWR, Radnor, Pennsylvania, USA) coated with 10% Matrigel^TM (BD Biosciences) were used as a barrier for invasion. As a chemoattractant, 2.5 ml of 3% FBS or 10% FBS was placed in the lower chamber. The cells were incubated at 37°C for 24 h and analyzed for cell invasion.
The cells on the lower side of the insert membrane were fixed with pre-chilled (4°C) methanol and stained with crystal violet solution (0.1%, Sigma®). The images were acquired under inverted light microscopy with phase contrast (Nikon ECLIPSE Ti, Melville, New York, USA) and processed using Adobe® Photoshop® 7. For each well, the invaded cells in all visual fields observed at 200× magnification were manually counted. Prior to seeding the cells in the culture plate, cell viability was determined by exclusion of trypan blue dye. The experiments were conducted three times with comparable results.