Transwell chambers 3422

Antibodies for SCYL1 (A6735) and Actin (A5441) were purchased from Abconal Technology (Wuhan, China) and Sigma-Aldrich (St. Louis, MO, USA), separately. Anti-Mouse (31,430) and anti-Rabbit (31,460) HRP-conjugated secondary antibodies were from ThermoFisher Scientific (Waltham, MA, USA). The shRNA oligos for SCYL1 were produced by Sangon Biotech Company (Shanghai, China). Transwell chambers (3422) were purchased from Corning Inc (Corning, NY, USA). Puromycin was purchased from Biotopped (Beijing, China). The DAB kit for IHC was from Zhongshan Jinqiao Biotechnology (Beijing, China). The SABC-POD kit and Mayor’s hematoxylin were purchased from Boster Biological Technology (Wuhan, China). Human breast cancer cell lines MDA-MB-231 and MCF-7 were obtained from ATCC (Manassas, VA, USA).

Corning Transwell chambers 3422 and 354,480 were used for the assessment of migration and invasion, respectively, using the experimental procedure reported in the literature [12 (link)]. Briefly, after 24 h of starvation, the cells were seeded in a Transwell chamber (100,000/well). After 48 h of culture, the cells were fixed with methanol, stained with crystal violet, and observed and photographed under a microscope. The number of cells that crossed the membrane was determined.

Transwell migration and invasion assays were assessed with Transwell Chambers 3422 (Corning, Kennebunk, USA). For a migration assay, 3 × 10⁴ cells in 200 μL serum-free medium after transfection for 24 h were seeded in the upper compartment of the chamber. We added 600 μL complete medium containing 10% FBS were added into the lower chamber. For an invasion assay, the upper surface of polycarbonic membranes was covered with a 60 μL matrigel (Becton-Dickinson, Franklin Lakes, NJ, USA) and incubated for 2 h at 37°C. The subsequent operations were similar to the migration assay. After incubation for another 24 h, the cells on the upper surface were removed with cotton swabs. Then the cells that had invaded into the microporous membrane were washed three times with PBS and fixed with 4% paraformaldehyde solution for 30 min, air dried, and stained with 0.1% crystal violet (Google biotechnology, Wuhan, China) for 20 min. Finally, the cells were observed with a microscope (Olympus BX51) and images were captured as well.