Polycarbonate membrane insert

Migration and invasion assays were performed using a 24-well migration and invasion plate containing a polycarbonate membrane insert (Cell Biolabs, San Diego, CA, USA). Media containing 10% fetal bovine serum (FBS) (500 μL) was added to the bottom chamber of the invasion or migration plate. Cells were suspended at a concentration of 1.0 × 10⁶ cells/mL in serum-free media. The cell suspension (300 μL) was added to the top chamber, treated directly with different concentrations of ramalin (12.5 and 25 μg/mL) and maintained in a cell culture incubator for 48 h. The cells were stained using a Cell Stain Solution (400 μL) and photographed.

The Boyden Chamber transwell migration assay was used to determine the migration rate towards MCP-1 upon LY86 KD [51 (link)]. A polycarbonate membrane insert with a 5 μM pore size (Cell Biolabs) was placed in a well of a 24-wells plate filled with 500 μl Accell delivery medium supplemented with 0.5% BSA with or without 100 ng/ml human MCP-1 (Prospec). The insert was filled with 100 μl Accell delivery medium supplemented with 0.5% BSA and 100,000 THP-1 monocytes treated for 72h with LY68 siRNA or Green non-targeting siRNA. Cells were placed in a humidified incubator with 5% CO2 at 37 °C. After 3h, the number of migratory cells was quantified in the bottom well using a hemocytometer. Significance was calculated using an unpaired t-test.