Biocoat matrigel invasion chambers with 8.0 m pet membrane

Corning® BioCoat® Matrigel® Invasion Chambers with 8.0 µm PET Membrane were used to perform invasion assay. Membranes were rehydrated for 2 h in a 5% CO2 incubator. 1 × 10⁵ cells were seeded in the upper chamber in FBS-free medium and the lower chamber was filled with complete medium supplied with 10% FBS. After 24 h, cells in the upper chamber were removed, and invaded cells embedded in the membrane were fixed and coloured with crystal violet solution (0,2% crystal violet, 2% EtOH, H₂O) for 10 min. Stained cells were eluted with 1% SDS, rocking for 10 min, and quantified at 570 nm absorbance. Experiments were repeated four times.

The cell invasion assay was performed using BioCoat Matrigel Invasion Chambers with 8.0 µm PET Membrane (Corning) according to the manufacturer’s instructions. Briefly, 7.5 × 10⁵ 4T1 and 6DT1 cells were seeded onto the top well containing DMEM with 0.5% serum. In the bottom well, DMEM with 10% serum was used as a chemoattractant. Cells were incubated in a 37 °C incubator for 24 and 48 h for 6DT1 and 4T1 cells, respectively. After incubation, noninvaded cells were removed from the top well using cotton tips. Cells that had invaded into the Matrigel were then fixed with methanol and stained with 0.05% crystal violet. Matrigel membranes containing invaded cells were then cut and mounted onto glass slides with Vectashield mounting medium (Vector Laboratories). Images of entire membranes were captured as segments and then stitched using an EVOS FL Auto 2 microscope (Invitrogen). Image analysis was performed using the Image-Pro Premier 3D (Media Cybernetics) software. Percent cell invasion was calculated using crystal violet intensity per membrane area.

Migration and invasion assays were performed using the Falcon® Permeable Support for 24-well Plate with 8.0 µm Transparent PET Membrane and Corning® BioCoat™ Matrigel® Invasion Chambers with 8.0 µm PET Membrane (Corning, NY, United States) according to the manufacturer’s procedure. Cell numbers and incubation times were as follows: migration assays, 2 × 10⁴ cells per chamber and 24 h; invasion assays, 5 × 10⁴ cells per chamber and 48 h. We used 10% FBS as a chemoattractant. After incubation, the migrated or invaded cells from upper chamber to the opposite side of the PET Membrane were fixed using 100% methanol at room temperature and stained with a Giemsa stain. The number of migrated or invaded cells was counted under a microscope.

Migration and invasion assays were performed using a modified Boyden chamber (6.5 mm Transwell with 8.0 µm Pore Polycarbonate Membrane Insert; Corning) and a Matrigel-coated modified Boyden chamber (BioCoat Matrigel Invasion Chambers with 8.0 µm PET Membrane; Corning), respectively as described previously (Kurayoshi et al., 2006 (link); Matsumoto et al., 2014 (link)). In the standard conditions, S2-CP8 cells (2.5 × 10⁴ cells) were seeded in the upper side of Boyden Chamber. In GFP-expressing S2-CP8 cells, after 4 h (migration assay) or 24 hr (invasion assay, except for Figure 6E) incubation with control ASO, 122 cells (average) and 126 cells (average), respectively, were observed in the lower side chamber in the one field of view under fluorescence microscope (BZ-9000, Keyence, Osaka, Japan) using a 10 x air objective. In Figure 6E, cells were observed after 20 hr incubation with ASO. Migration and invasion rates of cells expressing ARL4C, IQGAP1, and MMP14 mutants were calculated as the percentages of the same cells transfected with control ASO or siRNA.