Fluorescence‐based quantitative scratch wound healing assay demonstrating the role of MAPKAPK‐2/3 in fibroblast migration

Abstract

The scratch wound healing assay is a sensitive method to characterize cell proliferation and migration, but it is difficult to be quantitatively evaluated. Therefore, we developed an infrared fluorescence detection‐based real‐time assay for sensitive and accurate quantification of cell migration in vitro. The method offers sensitivity, simplicity, and the potential for integration into automated large‐scale screening studies. A live cell staining lipophilic tracer—1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethyl indotricarbocyanine iodide (DiR)—is used for accurate imaging of wound closure in a simple 96‐well scratch assay. Scratches are made on prestained confluent cell monolayers using a pipette tip and scanned at different time intervals using a fluorescent scanner. Images are analyzed using Image J software and the migration index is calculated. Effect of cell number, time after scratch and software settings are analyzed. The method is validated by showing concentration‐ and time‐dependent effects of cytochalasin‐D on fibroblast migration. Using this assay, we quantitatively evaluate the role of the MAPK‐activated protein kinases MK2 and MK3 in fibroblast migration. First, the migratory phenotype of MK2‐deficient MEFs is analyzed in a retroviral rescue model. In addition, migration of MK2/3‐double‐deficient cells is determined and the ability of MK3 to rescue cell migration in MK2/3‐double‐deficient fibroblasts is demonstrated. Cell Motil. Cytoskeleton 2009.