Matrigel coated transwell invasion chambers

BON1/HA-Bcl-xL/TR-shCtBP2 and BON1/HA-Bcl-xL/TR-shRLuc #713 cells were treated with 1 μg/ml (before sorting) or 0.5 μg/ml (after sorting) doxycycline (Clontech Laboratories, Inc., 631311) for 96 hours, and 2 × 10⁵ of cells were seeded in Matrigel-coated transwell invasion chambers (Corning, 354480) with DMEM containing 1% FBS, 0.4 mg/ml G418, 0.5 μg/ml puromycin, 0.2 mM L-glutamine and 100 units/ml penicillin and 100 μg/ml streptomycin. The lower chambers were filled with DMEM containing 10% FBS, 0.4 mg/ml G418, 0.2 mM L-glutamine and 100 units/ml penicillin and 100 μg/ml streptomycin. Fifty thousand cells per cell line were seeded in 1% FBS, 0.4 mg/ml G418, 0.5 μg/ml puromycin, 0.2 mM L-glutamine and 100 units/ml penicillin and 100 μg/ml streptomycin per well of 24-well dish for the proliferation controls. After 24 hours of incubation, cells on the opposite side of the chambers were fixed in 4% paraformaldehyde in PBS for 10 min, washed with PBS−/− for 5 min, stained with 0.1% crystal violet for 30 min and counted in 8 fields under 20X magnification.