In vitro trophoblast migration and invasion assessment
The migration function of HTR8 and HIPEC65 cells was examined in Boyden chamber with an 8-μm pore size (Corning-Costar)(Chen, 2005). The cells were seeded into the top chamber, and were allowed to migrate toward the bottom chamber containing conditioned media in which cortisol (10-6M) was added. After 24h incubation, cell moved to the underside of the membrane were stained with DAPI (Beyotime Biotechnology). The number of the cells on the membrane from 3 random fields at ×200 magnification was counted under the microscope. In some cases, scramble siRNA and 11β-HSD2 siRNA mixed with LipofectamineTM3000 were added into the cells in order to knockdown of 11β-HSD2. After 12h incubation, the culture media were changed into fresh media containing cortisol (10-6M). After incubation for 24h, cell moved to underside of the membrane were stained with DAPI. The number of the cells on the membrane was counted as described above.
Invasion function was assessed by the ability of cells to digest and invade the Matrigel-coated 8 μm pore size polycarbonate membrane Transwell inserts (Corning-Costar) as described previously (Yang et al., 2012). Briefly, HIPEC65 or HTR8 cells seeded in the inserts and treated with cortisol for 24h. Noninvaded cells on the top of the filter were scraped off using a cotton swab, and cells were fixed and stained with DAPI for microscopic analysis. Invaded cells from 3 random fields at ×200 magnification were counted under the microscope. In some cases, cells were treated with scramble siRNA and 11β-HSD2 siRNA mixed with LipofectamineTM 3000. After 12-h incubation, the culture media were changed into fresh media containing cortisol (10-6M). Then the cells were assessed for invasion activity.