HaCaT Cell Migration Assay in Differentiation Conditions

HaCaT cells were first cultured in H-Ca medium and maintained in an incubator under 5% CO₂ at 37 °C. The short-term cell culture was used for the HaCaT cell migration assay, with incubation for 48 h in L-Ca medium. After 48 h culture in L-Ca medium, involucrin expression was minimal, similar to cells cultured in undifferentiated conditions. A cell migration assay was performed using a transwell chamber plate (6.5 mm D.I., 8.0 μm pore size, polycarbonate membrane; Transwell Permeable Supports 3422, Corning Inc., New York, NY, USA) as previously described [12 (link)]. Cells subjected to short-term cell culture as described above were seeded at a density of 2 × 10⁴ cells/well in the upper chamber supplemented with 200 μL H-Ca medium, while 750 μL H-Ca medium with or without experimental reagents (Sema3A, kCer, or histamine) was added to the lower chamber compartment. After 48 h incubation at 37 °C, the unmigrated cells in the upper chamber were gently removed with a cotton swab, and the cells that had migrated to the lower compartment of the membrane were fixed with cold absolute methanol for 10 min and stained with GIEMSA’S AZUR EOSIN Methylene Blue solution (109,204, Merck, Darmstadt, Germany) as described previously [17 (link)].