3 μm pore size transwell inserts

A 6-well plate and 3 μm pore size transwell inserts (Corning) were used to establish the co-culture systems as reported before^{54 (link)}. Bel7404/LM3 cell lines were seeded in a prepared 6-well plate with a number of 5 × 10⁴ cells/well. A number of 5 × 10⁴ cells/well of hMSCs were seeded in the transwell inserts located in neighboring wells. After cells were attached to the wall firmly, the transwell inserts with hMSCs were moved to the wells containing Bel7404/LM3. The Bel7404/LM3 in this coculture system was regarded as co-culture groups. In control groups, both plate wells and transwell inserts were seeded with Bel7404/LM3. After incubation for 48 h, the co-cultured cells (Bel7404/LM3 cells) were harvested. After being washed with PBS, cells were fixed with 70% ice-cold ethanol, incubated with Cell Cycle Staining Kit (BD Biosciences, San Jose, CA, USA) for 30 min in the dark, and analyzed by flow cytometry.

We established the coculture system through 6-well plate and 3 μm pore size transwell inserts (Corning). The SMCs (5 × 10⁵ per well) were seeded in 6-well plate and the same quantity of different treated ECs were seeded in the transwell inserts located in adjoining wells. The transwell inserts with ECs were moved to the wells containing SMCs when cells attached to the wall firmly (24 h). After another 48-h culture, SMCs were harvest for western blot. Similarly, we set up another co-culture system for migration experiments, 24-well plate and 8 μm pore size transwell chambers (Corning) were used instead, 5 × 10⁴ different treated ECs per well were seeded in 24-well plate and the same number of SMCs were seeded in up chambers adjacently. After cells attached firmly, the transwell chambers with ECs were moved to the wells containing ECs. Forty eight hours later, chambers were taken out for migration experiments.