Transwell Assay for Cell Migration

To further assess the effect of HSR2104 on LPS-stimulated cell mobility, transwell migration assays were performed as described previously [22 (link),23 (link)]. BV2 cells were seeded into the inserts (6.5 mm in diameter with an 8.0-μm pore size) of a Costar transwell system (Corning Inc., Kennebunk, ME, USA) at a density of 1 × 10⁵ cells/well and incubated at 37 °C for 24 h in DMEM supplemented with 10% FBS and 1% antibiotics. After incubation, the bottom wells were filled with HSR2104 at various concentrations with or without LPS (1 μg/mL) in serum-free media. Control cells were treated with serum-free media containing 1% DMSO instead. After 24 h of treatment, non-migrated cells were gently removed with a cotton swab and the migrated cells were fixed with 4% paraformaldehyde for 20 min, followed by permeabilization with methanol for 10 min prior to staining with 0.5% crystal violet for 10 min, as described previously [23 (link),60 (link)]. Four random fields from each well were captured and the numbers of cells migrated through the membrane were counted under a Nikon phase-contrast microscope (Nikon Instruments Inc., Melville, NY, USA). The cell migration was expressed as a percentage of the respective vehicle-treated control cells.