Transwell inserts in 24 well plates

The migration of the suitably treated cells was tracked using transwell inserts in 24-well plates (Corning, Shanghai, China). The cells were resuspended in serum-free medium and seeded in the upper chambers of the inserts at the density of 5 × 104 cells/well, and the lower chamber were filled with complete medium containing 10% FBS. After 24 h incubation, the unmigrated cells remaining in the upper chamber were wiped off with a cotton swab, and migrated cells on the underside of the filter were fixed with 4% paraformaldehyde and stained with crystal violet. The number of migrated cells were then counted under a microscope. For the invasion assay, membranes were coated with Matrigel (BD Biosciences, Shanghai, China) and the other steps were the same as in the migration assay.

Invasion assays were conducted using 8 μm-pore Transwell inserts in 24-well plates (Corning). We added 25 μl of growth factor-reduced phenol red-free Matrigel (BD), which was diluted in a 1:4 ratio with DMEM, to Transwell inserts on ice and incubated the inserts at 37 °C without disturbance. After 2 h, 3 × 10⁴ MCF-7 cells in 100 μl FBS-free DMEM were seeded in the upper chamber. The plates were divided into the following three groups: a group with CM-derived from CAFs with 10% FBS (CM-CAFs), a group with CM-derived from NFs with 10% FBS (CM-NFs) and a control group with DMEM and 10% FBS. After 48 h of incubation at 37 °C and 5% CO₂, the cells remaining in the upper chambers were removed with a cotton swab, and the inserts were fixed in cold 4% formaldehyde for 5 min and stained with 5% crystal violet for 5 min. Then, the cells that had invaded to the lower surface of the membranes were counted from six random fields of views with an inverted microscope at 400× magnification. The assay was performed in triplicate.