Fluorescence-based quantitative scratch wound healing assay demonstrating the role of MAPKAPK-2/3 in fibroblast migration.
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.
Menon
Manoj B
MB
Institute of Biochemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, Hannover, Germany.
Ronkina
Natalia
N
Schwermann
Jessica
J
Kotlyarov
Alexey
A
Gaestel
Matthias
M
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002232:Carbocyanines; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D005453:Fluorescence; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling; D014945:Wound Healing; D000099190:MAP-Kinase-Activated Kinase 2
D002232:Carbocyanines; D047908:Intracellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases; D000099190:MAP-Kinase-Activated Kinase 2; C098416:MAP-kinase-activated kinase 3