Quantitative Migration Assay of ESdM Cells

Quantitative migration assays were carried out using 8-μm pore FluoroBlock migration plates (Calbiochem; Darmstadt, Germany) as described previously [23 (link), 24 (link)]. ESdM cells were loaded with 5-mM Calcein-AM (Life Technologies) for 45 min at 37 °C and washed prior to seeding at 50,000 cells/well in the upper chamber of the tissue culture insert. CX3CL1 (10 ng/ml) was added to the lower chamber to stimulate migration. The number of migrated cells was counted using an inverted fluorescence live cell imaging system (Carl Zeiss MicroImaging; Thornwood, NY). Each experiment was performed in triplicate, and each experimental well was imaged five times in different locations, and the results were expressed as an average of the total number of migrated cells in response to chemoattractant under each experimental condition. The images were analyzed with AxioVision version 4.7 software (Carl Zeiss MicroImaging) and with National Institutes of Health ImageJ version 1.42 software (http://rsbweb.nih.gov/ij/) as described [25 (link)].