Quantifying Breast Cancer Cell Transmigration

Transmigration of transfected or control MDA231 and MDA231-BrM2 cells was assessed as previously described.13 (link),15 (link) Briefly, pre-transfected MDA231 and MDA231-BrM2 cells were labeled with 5 μM of CellTracker™Green CMFDA fluorescent dye (Thermo Fisher Scientific, USA, cat# C2925) as per the manufacturer’s protocol. Then 5×10⁴ labelled BC cells were gently added to the apical chambers of tight hCMEC/D3 monolayers. BC cells were allowed to transmigrate to the basal compartment containing complete growth medium for 24 hours. The remaining cells on the upper chamber were gently removed using a cotton swab, and BC cells that transmigrated to the basal compartment were lysed with RIPA 1× buffer for fluorescence quantification at a wavelength excitation/emission of 492/517 nm using a Varioskan Flash microplate reader (Thermo Fisher Scientific).