24 well transwell insert system

Cells at 1 ×10⁵ cells/well in 300 μl of serum-free medium were seeded into the upper chamber of a 24-well Transwell insert system (BD Biosciences, San Jose, CA, U.S.A.) with an 8-μm pore size polyethylene terephthalate membrane and 600 μl of 10% FBS-containing medium as a chemoattractant was added to the lower chambers. After 24-h incubation at 37°C in a 5% CO₂ incubator, non-migrated cells were removed from the upper side of each insert with cotton swab, while the migrated cells on the bottom surface of the insert were carefully washed with PBS, fixed in methanol for 10 min, and then stained with 0.5% Crystal Violet for 20 min. Migrated cells were counted with an inverted microscope at a magnification of ×200 in ten random fields in each well and migration rate was expressed as the percentage of migrated cells compared with that of the control group, which was set as 100%. Each experiment was performed in triplicate.

The migration ability of HTR-8/SVneo trophoblast cells was examined with a 24-well Transwell insert system (8 μm pore size, BD Biosciences). HTR-8/SVneo cells were seeded in the upper chamber of the insert (2 × 10⁴ cells/well), which contained 300 μL medium (DMEM/F12 medium with 5% FBS and 1% penicillin and streptomycin solution). The lower chambers were filled with the following: (1) 800 μL condition medium (0, 25, 50, or 100% hUCMSC or hFF supernatant in standard medium) or (2) 800 μL standard medium with 0, 2.5, 5, or 10 × 10⁴ hUCMSCs or hFF cells/well. After 16 hours in culture, 80 μL fluorescent stain (calcein-AM) was added to each chamber and incubated for 30 minutes. The labeled cells were observed and photographed with a fluorescence microscope (Nikon, Japan).