8 μm pore cell culture inserts

The migration of PANC-1 cells was assayed in 24-well plates with 8 μm pore cell culture inserts (BD, Franklin Lakes, NJ, USA). Five hundred microliters of RPMI medium supplemented with 10% FBS was added to each well, and 3 × 10⁴ cells were seeded into each insert. The cells were incubated with human IgG, CTX, and M-CTX-Fc, in a range of 0–300 nM in RPMI medium supplemented with 1% BSA at 37°C. After 48 h of culture, the insert chambers were removed and adherent cells on the bottom of each well were counted under microscope. The number of migrated cells was normalized to the number of adherent cells in the absence of human IgG, CTX, and M-CTX-Fc.

The invasiveness of HTR8/SVneo cells was examined with a Matrigel invasion assay using 8 μm-pore cell culture inserts (BD Biosciences) pre-coated with Matrigel (125 ng/ml; BD Biosciences) and inserted in a 24-well plate described previously (345 (link)). After transfection with ZNF554 or scrambled siRNAs for 24 h, 2 × 10⁵ cells were plated in the upper chambers in a serum-free RPMI-1640 medium, whereas an RPMI-1640 medium supplemented with 10% FBS was added to the lower chambers. After incubation for 48 h in 2, 8, or 20% O₂ concentrations, cells on the Matrigel side of the membranes were removed by cotton swab, and the membranes were processed as in the migration assay. Comprehensive images taken using the Leica TC5 SP5 spectral confocal system were quantified using Image-Pro Plus 6.2 (Media Cybernetics, Inc.). The experiment was performed in triplicate (Figure 10).

HBMECs were starved in SF EC medium (1% L-Glut, 1% PEST) O/N. Cells (4 × 10⁴) were added in SF media to the top chamber of 8-μm pore cell culture inserts (BD Biosciences) placed in a 24-well plate. THP-1 cells were differentiated in the top chamber of 8-μm pore cell culture inserts for 48 h, followed by starvation in SF medium for 48 h. Cells were incubated at 37 °C for 6 h to allow cell migration toward SF medium, or different CM (diluted 1:5 with SF medium for HBMECS and undiluted for THP-1 cells) collected from U-87 MG and GL261 cells, as indicated in the respective figure legend. Migrated cells attached to bottom membrane were fixed with 4% paraformaldehyde (PFA) for 15 min, stained with crystal violet, and counted from pictures taken under the microscope (Axiovert 40C, 4× objective; Carl Zeiss).