Boyden chamber transwells

Boyden chamber transwells (Corning, Corning, NY, USA, 3422, pore size: 8 μm) coated with 70 μl diluted Matrigel (1 volume Matrigel: 2 volumes ice-cold serum-free media) were used to measure in vitro invasive capacity, as previously described [27 ]. 2 × 10⁴ modified β6-1089 or N-1089 cells were plated in serum-free media in the lower chamber. 3 × 10⁴ MCF-7, MDA-MB-231or SUM159 breast cancer cells were seeded onto the Matrigel-coated insert. Invasion assays were carried out at 37 °C over 24 hours, or 48 hours for MCF-7 cells. Invaded cells were harvested with 10× trypsin/EDTA (PAA Laboratories, L11-003) from the underside of the transwell and counted with a CASY counter (Schärfe System, Reutlingen, Germany).

Cell growth was assessed using the sulforhodamine B assay as described [30 (link), 48 (link)]. Wound healing assay was conducted to assess cell migration in vitro as described [49 (link)]. Briefly, sub-confluent monolayer cells in 24-well plates were scratched with a sterile 200 μl pipette tip to create a wound line in the middle of the well before transfection with saRNAs as indicated in the figures. Cell migration was monitored for 3 days.
Boyden Chamber transwells from Corning (Tewksbury, MA) were used to assess cell migration in vitro as described [50 (link)] with modifications. Briefly, 2 × 10⁴ cells were transfected with the saRNAs as indicated in 6-well plates and transferred into the upper chambers. Migrated cells in the lower chamber were stained with fluorescent dye Hoechst-33342 (final concentration at 5 μg/ml) and counted at 3 days after transfection.

Cell migration and invasion assays were assessed using 8.0 µm pore size Boyden-Chamber transwells (Corning). For cell invasion assays, chambers were first pre-coated with 20 µL (1:10 dilution) of low serum Matrigel (Cultrex) and allowed to set for 60 min. Cells were serum starved overnight and then seeded into the upper chamber (2 × 10⁴ cells) in 500 µL serum free medium. 500 µL medium with 10% FCS was added to the lower chambers. After 24-h, non-migrated/non-invasive cells in the upper chamber were removed using a cotton swab and migrating/invasive cells were fixed in 10% buffered formalin and stained with 0.1% crystal violet. The number of migrated/invaded cells was analysed using the ImageJ software.