Fluoroblok cell culture insert

150,000 cells/cm² were seeded in the top part of Corning® FluoroBlok™ Cell Culture Inserts in DMEM serum-deprived medium for 24 h. Medium in the bottom chamber was then replaced by 10% FBS DMEM medium to induce cell migration. After 16 h, cells were labeled with the fluorescent Calcein AM dye (ThermoFisher Scientific) in Hank’s Balanced Salt Solution for 2 h. Bottom Fluorescence intensity, corresponding to post-migratory cells was measured using a FlexStation3 microplate reader at λ 495 nm.

For migration assays, Corning^® FluoroBlok™ Cell Culture Inserts with a pore size of 8 μm combined with 24‐well cell culture plates were used. Before start of the migration assay, the cells were serum‐starved overnight in RPMI with 1% BSA. Cells were then stained with CellTracker™ Red CMPTX dye (Thermo Fisher) at a final concentration of 10 µM for 30 minutes in serum‐free RPMI without phenol red. Cells were pelleted and again incubated in serum‐free RPMI for 30 minutes without phenol red to remove the surplus dye. Cells were seeded in duplets onto the insert membrane at a density of 3 × 10⁵ cells/300 µL per insert. The bottom compartment was filled with 1.0 mL of the same medium. After 30 minutes rest period, cells were placed into wells containing the specified chemoattractant and were allowed to migrate through the membrane for 48 hours. Migration was quantified from the bottom of the wells by fluorescence measurement using the CLARIOstar microplate reader (BMG; Extinction: 577 nm; Emission: 607 nm) every 10 minutes within the first two hours and then every 30 minutes for 46 hours. Cell migration was displayed as signal compared to the measurement at 4 hours.

The migration of the endothelial cells towards the NF membranes through 8 µm diameter pores was analysed using a transmigration assay setup. The membranes were placed on 24-well plates with a low-supplemented EGM-2 medium. FluoroBlok cell culture inserts (Corning, New York, NY, USA) were placed in each well and 20,000 HSVEC were seeded in the inserts. After 4 h in the cultures, the inserts were washed with PBS and fixed with 4% paraformaldehyde in PBS for 15 min at RT. The cells were then stained with a combination of the Texas Red C₂-maleimide cell membrane dye (1.7 µg/mL in PBS, ThermoFisher Scientific, Waltham, MA, USA) and the Hoechst 33,258 nuclear dye (5 µg/mL of PBS) for 1 h at RT. Images of the cells that crossed the insert membrane were captured under an Olympus IX-71 epifluorescence microscope equipped with a DP80 digital camera (both from Olympus, Tokyo, Japan).

The PET membranes (8 µm pore size) of FluoroBlok™ cell culture inserts (351152, Corning, NY, USA) were coated with 90 µL of diluted Matrigel (0.3 mg/mL) (356234, Corning), and incubated at 37 °C for 2–3 h until solidified. Next, 1 × 10⁴ of 4T1, BT20 or MDA-MB231 cells suspended in serum-free DMEM media were seeded on the solidified Matrigel layer. Then, 700 µL of chemo-attractant medium (DMEM, 1% P/S and 10% FBS) was added to the lower chambers (353504, multiwell 24 well companion plate, Corning), and the plate was incubated in a 37 °C incubator. After 24 h of incubation, the insert bottoms were dipped in 1× PBS and stained in diluted Calcein AM (354217, Corning) in PBS for 10 min. Fluorescence images of invaded cells were captured with an EVOS inverted microscope, and analysis was done with ImageJ software.

We performed cell migration assays using Corning FluoroBlok cell culture inserts and according to manufacturer's protocol. Briefly, fluorescently-labeled cells were allowed to migrate for up to 48 hours, and the percentage of cells migrated into the plane of view was calculated. Cells that have migrated through the membrane were detected using a bottom-reading fluorescence microscope. Three areas of view per biological replicate (n = 2) were counted at 4, 18, 24, and 48 hours postseeding Student t tests were performed at each timepoint.