Transwell assay insert

The methods used for migration assay and invasion assay are almost the same. We put transwell assay inserts (Millipore, Billerica, MA, USA) in a well of 24-well plates. However,the difference is the membrane in the upper chamber of transwell assay in invasion assay is Matrigel-coated membrane(BD Biosciences) while the one in migration assay is only a normal membrane. In the experiment, firstly, we set 500ul serum-free RPMI1640 at bottom chamber. Following this, we seeded 10,000 cells in 200ul RPMI1640 with 10% FBS at upper chamber. After 24 h or 48 h, we used methanol to fix the cells within the membrane and stain them with crystal violet. Our final task is to observe these cells by microscope.

The methods used for the migration assay and invasion assay were similar. We placed transwell assay inserts (Millipore, Billerica, MA, USA) in a 24-well plate. However, the difference between the methods lies in the type of membrane: The membrane in the upper transwell chamber for the invasion assay was a Matrigel-coated membrane (BD Biosciences), while that for the migration assay was a normal membrane. In the experiment, first, we placed 500 μl of serum-free RPMI 1640 with 10% FBS in the bottom chamber. Following this, we seeded 10,000 cells in 200 μl of RPMI 1640 in the upper chamber. After 24 to 48 h, we used methanol to fix the cells within the membrane and stained them with crystal violet. Finally, the cells were observed by microscope.

Lung cancer cells were transfected with 50 nM si-ANRIL or si-NC. After 24 h, transfected cells were harvested. In migration assay, transfected cells (1x10⁵) were plated in the top chamber of Transwell assay inserts (Millipore) with a membrane containing pores with 8 mm diameters in 200 ml of serum-free RPMI1640. Assays were conducted in triplicate. Inserts were then placed into the bottom chamber wells of a 24-well plate containing RPMI1640 with 10% FBS as a chemoattractant. After 24 h of incubation, remaining cells were removed from the top layer of the insert by scrubbing with a sterile cotton swab. Invading cells from the bottom surface were stained with 0.1% crystal violet prior to being examined, counted and photographed using digital microscopy. Cell numbers were calculated in five random fields for each chamber, and the average value was calculated. In invasion assay, transfected cells (4x10⁵) were plated in the top chamber with a Matrigel-coated membrane. Bottom chambers were filled with conditioned medium. After a 48 h incubation period, the number of migrated cells on the lower side of the membrane was counted as described previously.

For migration assays, transfected cells (40,000 cells in 100ul per well) were plated in the upper chamber of trans-well assay inserts (8 mm pores, Millipore, Billerica, MA) containing 200ul of serum-free RPMI1640 media. The lower chambers were filled with RPMI1640 containing 10% FBS. After 24 h of incubation, cells on the filter surface were fixed with methanol, stained with crystal violet, and photographed. Migration was assessed by counting the number of stained cell nuclei from 5 random fields per filter in each group.
For invasion assays, transfected cells (40,000 cells in 100ul per well) were plated in the top chamber with a matrigel-coated membrane (BD Biosciences) in 300ul serum-free RPMI1640. The bottom chambers were filled with RPMI1640 containing 10% FBS. Invasion was determined after 48 h incubation.
For wound healing assay, cells were seeded and transfected on six-well plates with si-SNAP23or si-NC, then an artificial scratch wound on a confluent monolayer of A2780 or SKcells was created with a 200-μl pipette tip. Serum-free medium was added for a further 24-h, and cells were imaged 24 h later. Each experiment was repeated three times.

The methods used for the migration assay and invasion assay were similar. We placed transwell assay inserts (Millipore, Billerica, MA, USA) in a 24-well plate. However, the difference between the methods lies in the type of membrane: The membrane in the upper transwell chamber for the invasion assay was a Matrigel-coated membrane (BD Biosciences), while that for the migration assay was a normal membrane. In the experiment, we first placed 500 μl of serum-free RPMI 1640 with 10% FBS in the bottom chamber. Next, we seeded 10,000 cells in 200 μl of RPMI 1640 in the upper chamber. After 24–48 h, we used methanol to fix the cells within the membrane and stained them with crystal violet. Finally, the cells were observed by microscopy.