Matrigel coated transwell membrane filter inserts

Human RCC cells were seeded in 96-well plates with 3x10³ cells/well for XTT assays. After 72 h, cell proliferation was determined using a Cell Proliferation Kit II (Roche Diagnostics GmbH, Mannheim, Germany) as described previously [40 (link)].
Cell migration activity was evaluated with wound healing assays. Cells were plated in 6-well plates at 2x10⁵ cells per well, and after 48 h of transfection the cell monolayer was scraped using a P-20 micropipette tip. The initial (0 h) and residual gap lengths 24 h after wounding were calculated from photomicrographs.
Cell invasion assays were performed using modified Boyden chambers consisting of Matrigel-coated Transwell membrane filter inserts with 8-μM pores in 24-well tissue culture plates (BD Biosciences, San Jose, CA, USA). 48 h following transfection, the cells were seeded in the upper chamber of 24-well plates at 1x10⁵/well with serum-free RPMI 1640 medium. RPMI containing 10% exosome-depleted FBS in the lower chamber served as the chemoattractant, as previously described [42 (link)]. 24 h after seeding, the cells that had passed through the pores and attached to the surface of the chamber were stained by Diff-Quick (a modified Giemsa stain) (Richard Allan Scientific, San Diego, CA, USA) and counted from photomicrographs.

To evaluate cell proliferation, we used XTT assays. BOY and T24 cells were seeded in 96‐well plates (1 × 10³ cells/well) with 100 μL medium per well. We determined the extent of cell proliferation 96 h after seeding using a Cell Proliferation Kit II (Roche Diagnostics GmbH, Mannheim, Germany). When using cisplatin, gemcitabine, or Compound 3144, we added 10 μL of the stock solution, adjusted to 10‐times the target concentration. Inhibition data were used to calculate the half‐maximal inhibitory concentration (IC₅₀) values using nonlinear, four‐parameter, variable slope equation software (graphpad prism ver. 8.00 for Windows; GraphPad Software, San Diego, CA, USA). Wound healing assays were used to assess cell migration activity. Cells (2 × 10⁵ cells/well) were plated in 6‐well plates, and after 48 h of incubation, the cell monolayer was scraped using a P‐1000 micropipette tip. The initial gap length (0 h) and the residual gap length 24 h after wounding were calculated from photomicrographs. For cell invasion assays, we used modified Boyden chambers consisting of Matrigel‐coated Transwell membrane filter inserts with 8‐μm pores in 24‐well tissue culture plates (BD Biosciences, San Jose, CA, USA). The cells that passed through the pores and attached to the surface of the chamber were counted from photomicrographs.