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.

• Authors: Manoj B Menon, Natalia Ronkina, Jessica Schwermann, Alexey Kotlyarov, Matthias Gaestel
• Journal: Cell motility and the cytoskeleton (Cell Motil Cytoskeleton) Vol. 66 Issue 12 Pg. 1041-7 (Dec 2009) ISSN: 1097-0169 [Electronic] United States
• PMID: 19743408 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: (c) 2009 Wiley-Liss, Inc.

Chemical References

• Carbocyanines
• Intracellular Signaling Peptides and Proteins
• MAP-kinase-activated kinase 2
• MAP-kinase-activated kinase 3
• Protein Serine-Threonine Kinases

Topics

• Animals
• Biological Assay (methods)
• Carbocyanines
• Cell Movement (physiology)
• Cells, Cultured
• Embryo, Mammalian (cytology)
• Fibroblasts (cytology, physiology)
• Fluorescence
• Intracellular Signaling Peptides and Proteins (genetics, metabolism)
• Mice
• Protein Serine-Threonine Kinases (genetics, metabolism)
• Staining and Labeling
• Wound Healing