Chemotaxis Assay for KLS Cells

For chemotaxis assays, isolated KLS cells were suspended in migration medium comprised of RPMI-1640 medium, supplemented with 0.5% BSA, and then seeded in 8 µm pore transwell plates (Corning-Costar Corp). Murine SDF-1α (50, 100, or 200ng/mL; Sigma-Aldrich) or S1P (10 or 100nM; Sigma-Aldrich) was added to the lower chamber (specific concentration is indicated for each figure). KLS cells (1×10⁴ or 5×10⁴ cells) were placed in the upper well and incubated at 37°C for 4 hours and the number of cells in the lower wells was determined by flow cytometry. The migration index was calculated as the fold of increase in migration to the chemoattractant relative to baseline migration (without chemoattractant). Levels of SDF-1α and S1P were selected based on previous reports^{32 (link),33 (link)}.