Cell stain solution

Migration assays were performed similarly to Moses et al.^{59 (link)}. Transwell polycarbonate membrane filter inserts with 8 μm pore size (Cell Biolabs) were rehydrated and placed in 24-well tissue culture plates. MCF-7, T-47D, and SUM52PE cells were transduced with either AAMDC shRNAs or empty vector pLKO.1. The cells were serum-deprived overnight and added to the top chambers of the transwells. Ten percent serum-containing media was added to bottom chambers. The plates were next incubated at 37 °C for 24 h and then the culture media was aspirated. To visualize the migratory cells, the inserts were incubated with 400 μL cell stain solution (Cell Biolabs) for 10 min and pictures were captured using a cell culture inverted Leika light microscope to quantitate the number of cells that had passed through the porous membrane in 12 random fields. Results were expressed as average values relative to the empty vector pLKO.1 cells.

The CytoSelect 24‐well Migration Assays (Cell Biolabs, San Diego, CA, USA) were used to measure the migratory capabilities of OS and other cancer cell lines. 3 × 10⁵ cells grown in a serum‐depleted medium for overnight were added to the top chamber of a transwell. Then, the culture medium containing 10% FBS was added to the bottom well. After 4–6 h of incubation, migrated cells were stained by the Cell Stain Solution, which contains crystal violet. The numbers of migrated/invaded cells in five random fields under a microscope were counted in each condition/cell line, and the results were analyzed by two‐sample, two‐sided Student's t‐test. For the cell adhesion assay, 96 wells were coated with or without 10% Matrigel (BD Biosciences, San Jose, CA, USA), 10 µg·mL⁻¹ fibronectin, and 10 µg·mL⁻¹ collagen type I (Millipore, Burlington, MA, USA). Then, 1 × 10⁵ cells per well were plated and incubated for 30 min, and then washed twice with PBS. The cells were incubated with the Cell Stain Solution (Cell Biolabs) for 15 min, washed, and dried. One hundred microtiters of the Elution Buffer (Cell Biolabs) was added, and the absorbance at 520 nm was measured using the Multiskan FC Microplate Photometer (Thermo Fisher Scientific).

The upper surfaces of 8.0 µm Transwell supports (Greiner Bio-One) were coated with 20µl of growth factor reduced Matrigel (2.5 mg/ml; BD biosciences) for 1 h at 37° C. Excess Matrigel was removed and both chambers were allowed to equilibrate in plain DMEM at 37° C for 1 hour. Following equilibration, the bottom chamber was filled with 750 µl of DMEM/10% FBS containing inhibitors or DMSO control. 50,000 cells were re-suspended in 250 µl of DMEM/0.5% FBS containing inhibitors or DMSO control, plated in the upper chamber, and allowed to invade for 24 hours. Prior to fixation, cells that did not invade were removed from the upper surface of the membranes using a cotton swab. Membranes were fixed in ice-cold methanol for 10 minutes, stained with cell stain solution (Cell Biolabs), washed extensively with DDW, and allowed to dry overnight. Intact membranes were imaged using a Nikon Eclipse TS100 inverted microscope (10×, NA 0.25, air objective). For chemotactic migration assays, cells were plated in the upper chamber of Transwell supports without Matrigel coating and allowed to migrate for 24 hours followed by fixation and staining as above. Invasion index was calculated by subtracting values of chemotactic migration through un-coated membranes from values of chemotactic invasion through Matrigel-coated membranes.