Transwell cell culture plate

THP1 and SKGT EAC cell lines were obtained from the American Type Culture Collection. THP1 monocytes were cultured in RPM1 1640 containing 2 mM glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g l⁻¹ of glucose, 10% fetal calf serum, and 0.05 mMβ-mercaptoethanol. SKGT cells were grown in RPMI 1640 supplemented with 2% fetal calf serum, penicillin, and gentamicin. Cells were cultured in 100 mm cell culture dishes or 6/24-well cell culture plates. 12 h before the beginning of each experiment, cells were cultured in serum-free medium with antibiotics. To polarise THP1 with SKGT cell, a co-culture was done with Transwell cell culture plate (Corning Life Sciences, Corning, NY, USA). The insert with 0.4 μm pores was seeded with SKGT cells at 3 × 10⁶ as induced group and without any cells as control group. THP1 cells were plated in the bottom chamber at 2 × 10⁶. After 3 days culture, the insert was discarded, and the polarised THP1 cell was examined by microscopy and then used for RNA isolation or migration and invasion tests.

The lubricity properties of the lubricated gene-hydrogel patches were characterized by a high-temperature microdynamic wear tester (Optimal Instruments, USA). The prepared patches were placed on a sample table at a controlled temperature of 37°C, a friction frequency of 1 Hz, an amplitude of 4 mm, a load of 1 N and run for 1500 s, and the experimental data were recorded. Then, the patches were spread on the bottom of 24-well plates and inoculated with 208F cells, which were cocultured with the patches for 48 hours. The cytoskeletons and nuclei were stained using actin staining and DAPI staining, respectively, and the cell-spreading area was assessed. Then, 208F cells were inoculated in the lower chamber of a 24-well Transwell cell culture plate (Corning, USA), and the upper chamber was loaded with lubricated gene-hydrogel patches to coculture the cells and patches. The nuclei were stained with DAPI on days 1, 3, and 5 to detect siRNA release. Gene silencing efficiency was detected using qRT-PCR. The proliferation rate of each group of 208F cells was measured by CCK-8 assay according to the manufacturer’s protocol. The results were expressed using the proliferation rate by normalizing the mean absorbance value for each corresponding time point to the control.

PMNs (120 μl of 5 × 10⁶/ml) were added to 480 μl aEV, lysed aEV, sEV, apoEV sample or HBSS at 37°C in a linear shaker (0.18 g) for 45 min. aEV, lysed aEV, and sEV samples were prepared from 1.92 × 10⁷ cells, whereas apoEV samples were derived from 0.96 × 10⁷ cells. The pretreated PMN samples were placed in the wells of a 3 μm pore Corning, NY, USA transwell cell culture plate coated with 10% FBS. Every well contained 2 × 10⁵ cells. As a chemoattractant, 100 nM N-formylmethionyl-leucyl-phenylalanine was used. After 1 h incubation at 37°C, the transwell plate was centrifuged (Eppendorf 5810 R swing-bucket plate rotor, 3,220 g, 3 min, 4°C). Transmigrated cells were counted using an acid phosphatase assay^{37 (link)} in a plate reader (Labsystems iEMS Reader MF; Thermo Scientific).