6.5 nm transwell chambers with 8 m pores

This assay was performed to examine the modulation of OSCC cells during invasion by treatment with PL metabolites. Briefly, the membranes of 6.5 nm transwell chambers with 8 µm pores (Corning, United States) were coated with 50ul of gelatinous matrix Miogel (2.4 mg/ml) with type I collagen (0.8 mg/ml) (Corning, Cat # 354236) (Salo et al., 2015 (link))and incubated for 12–16 h. Then, the cells previously treated and non-treated with PL were plated in 200ul of DMEM/F12 medium without FBS, and subjected to the invasion assay according to Rodrigues AN et al. (2022) (link) (Rodrigues et al., 2022 (link)). The invasion profile was assessed by measuring the absorbance of the cell dye at 650 nm using an Epoch plate reader (BioTek).

Vertical migration assay was performed in 6.5 nm transwell chambers with 8 µm pores (Corning, NY, USA). Briefly, serum starved cells (8x10⁴ cells/well) were plated to the upper chamber in 200 µl of serum-free DMEM/F12, whereas the lower chamber was filled with 500 µl of DMEM/F12 supplemented with 10% FBS. After incubation of 24 h, nonmigratory cells in the upper chamber were gently removed with a cotton swab and cells that migrated to the bottom of the membrane were fixed and stained with a solution of 1% toluidine blue. After elution with 1% SDS solution, absorbance was measured at 650 nm in an ELISA reader (Bio-Rad Laboratories, USA).