Matrigel coated polycarbonate membrane insert

PDLSCs were plated at 3 × 10⁴ cells per well in the lower transwell chamber and cultured to 80% confluence in culture medium (α-MEM with 10% FBS and 1% antibiotic). The medium was replaced with an osteogenic medium containing 0, 0.01, 0.05, 0.25, 1.25 or 6.25 ng/ml IL-1β and cells were cultured for 24 h. An osteogenic medium with 0, 0.01, 0.05, 0.25, 1.25 or 6.25 ng/ml IL-1β and without PDLSCs used as a control. A total of 3 × 10⁴ RAW 264.7 cells were plated on a Matrigel-coated polycarbonate membrane insert (8.0 mm pores) in a transwell apparatus (Costar, Shanghai, China) and maintained in 100 μl of complete medium (α-MEM with 10% FBS and 1% antibiotic). Then, the inserts were washed with PBS, and the cells on the top surface of the insert were removed by wiping the surfaces with a cotton swab. The cells that migrated to the bottom surface of the insert were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet and then subjected to a microscopic inspection and cell count. The cells were counted under × 200 magnification. Every sample was counted in five randomly chosen fields and the values were averaged.

A total of 1 × 10⁵ RAW 264.7 cells were seeded on a Matrigel-coated polycarbonate membrane insert (8.0 µm pores) in a transwell apparatus (Costar) and maintained in 100 µl of complete medium (high-glucose DMEM with 10% FBS and 1% antibiotic). NP cells were also cultured in complete medium with or without 10 µg/ml LPS or 100 µM cordycepin in the lower chamber for 24 h. Then, the inserts were washed with PBS, and the cells on the top surface of the insert were carefully removed by using a cotton swab. The cells on the bottom surface of the insert were fixed with 4% paraformaldehyde for 10 min, followed by staining with 0.1% crystal violet for 20 min, and then subjected to an inspection and cell count via microscopy. The cells were counted under 200× magnification, and the counts of 5 randomly chosen fields were averaged for each sample.