Polyethylene terephthalate membrane filters

For tumor sphere formation and immune cell interaction with the Transwell co-culture system, confluent TAM-control or TAM-shSPP1/shSpp1 were placed in the upper insert (6.5 mm diameter with Polyethylene terephthalate membrane filters containing 0.4 μm pores, Corning, NY, USA). After induction, the inserts were moved into wells attached to Huh7 or preactivated T cells.

Cell migration assays were performed in 24‐well transwell plates with 8 µm polyethylene terephthalate membrane filters (Corning) separating the lower and upper culture chambers. THP‐1 were seeded in the upper chamber at 1 × 10⁶ cells per well in DMEM with 1% FBS. The bottom chamber contained DMEM with 10% FBS. THP‐1 cells were allowed to migrate for 24 h. After the incubation period, the filter was removed, and non‐migrant cells on the upper side of the filter were detached using a cotton swab. Filters were fixed with 4% formaldehyde for 15 min, and the cells located in the lower filter were stained with 0.1% crystal violet for 20 min and counted in three random fields.

In the colony formation assay, BC cells with the changed genes were diluted with single-cell suspension and we seeded 1000 or 2000 BC cells in each of the 6-well plates and incubated with 5% CO₂ at 37 °C for one week. The colonies were then stained with 0.04% crystal violet in 2% ethanol and counted.
Bladder cancer cell migration assay was conducted and 1000 or 2000 BC cells were seeded in a 24-well Transwell plate with 8 mm polyethylene terephthalate membrane filters (Corning). Cells were allowed to migrate for 24 h at 37 °C in a humidification chamber containing 5% CO₂. After incubation, the filter was removed and fixed with 4% formaldehyde for 15 min, followed by staining with 0.1% crystal violet for 20 min, and cells were counted.