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.