Keratinocytes transwell migration assay

Migration was assessed in a transwell assay with a polycarbonate membrane filter (Corning Costar, 6.5 mm diameter, 8 μm pore size). Briefly, 1 × 10⁵ cells were placed in the upper chamber of a transwell. After 6 hours cells remaining on the upper surface of the membrane were removed with cotton swabs and migrated cells on the lower surface of the membrane were stained with DAPI and counted by fluorescence microscopy. Assays were performed in triplicates.

In some cases, NHEK cells were grown in low glucose (5 mM) or high glucose (25 mM) medium for 5 days and conditioned medium (CM) from the last 48 hours was collected. 1 × 10⁵ cells were added to the upper chamber of a transwell chamber (Corning) in fresh medium or conditioned medium with or without activated MMP9 protein to measure the effect on keratinocyte migration. In some cases, conditioned medium in high glucose conditions was first collected from primary keratinocytes isolated from K14.Cre⁻.Foxo1 ^L/L mice and K14.Cre⁺.Foxo1 ^L/L mice. Then the cells were pre-incubated with indicated dose of MMP9 inhibitor (Millipore), and incubated in normal medium or conditioned medium collected above plus MMP9 inhibitor, which was added to the upper chamber of the transwell.