Matrigel coated transwell

Matrigel coated transwells (BD Biosciences) were prepared by soaking in serum-free media for 2 h at 37ºC in a 24-well plate. Subsequently, Caki-2 cells (5000/well) pre-transfected with either 25 nM siDCLK1 or siSCR for 48 h were seeded into each transwell in serum-free media in triplicate. Cell culture medium containing 10% FBS was added to the bottom of each well as chemoattractant and the cells were incubated for 22h at 37°C under 5% CO₂. Afterwards, a cotton swab was used to scrape non-invasive/migratory cells off the top of transwells and the remaining cells were fixed with 100% methanol, stained with 0.1% crystal violet, and allowed to dry. After drying all invading cells were counted from each transwell at 4x magnification. Cell number was normalized to siSCR control x 100 and results were reported as invasion (%siSCR Control).

PaCa cells were transfected with 50 nM of indicated siRNAs, and 72 h later, equal number of the treated viable cells (4×10⁴ cells), were seeded onto Matrigel-coated Transwells (with 8- µm pore size filters) in Matrigel invasion chambers (BD Biosciences, San Jose, CA). The number of cells that invaded the lower side of the membrane after 24 h was determined by counting cells in a minimum of four randomly selected areas. The experiments were performed in triplicate and the results were reported as mean of percentages of invasion ± standard deviation.

Control or matrigel coated transwells (BD Biosciences) were prepared by soaking in serum-free media for 2 h at 37°C in a 24-well plate. Subsequently, AsPC-1 cells (5000/well) were seeded into each transwell in serum-free media and treated with LRRK2-IN-1 or DMSO (vehicle) in duplicate. Cell culture medium containing 10% FBS was added to the bottom of each well as chemoattractant and the cells were incubated for 22 h. Afterwards, a cotton swab was used to scrape non-invasive/migratory cells off the top of transwells and the remaining cells were fixed with 100% methanol, stained with 1% toluidine blue/1% borax, and allowed to dry. After drying, the film from the transwell inserts was removed with a scalpel blade and mounted on slides. For each sample 5 fields were counted at 10X magnification. Percent invasion was calculated by dividing the number of invading cells (matrigel-coated inserts) by the number of migrating cells (control inserts) and multiplying by 100.

Uncoated Costar transwells (Corning Costar Co., Corning, NY) were used for migration assays and Matrigel-coated transwells (BD Biosciences, Franklin Lakes, NJ) used for invasion assays. Cells were serum starved overnight and then seeded into the upper chamber with serum-free RPMI 1640 medium. RPMI 1640 supplemented with 10% FBS was added into the lower chamber. Cells that had migrated across the transwell membrane were stained and quantified. For wound healing assay, the scratch was made across the cell monolayer using a sterile tip. The ability of cells to migrate was monitored at different time points using a light microscopy.