24-Well Haptotaxis Assays, Collagen I

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	Incubate for 4 hours in a cell culture incubator. Add 300μl of Lysis Buffer/CyQuantR GR dye solution

Protocol tips
Incubate for 4 hours in a cell culture incubator. Add 300μl of Lysis Buffer/CyQuantR GR dye solution

Publication protocol

HEK293T cells were cultured and transfected with plasmids encoding EphA2 and EphA2ΔSAM. Twelve hour following transfection, the cells were serum starved overnight. A cell suspension of 1×106 cells per ml was prepared in serum free medium containing 0.5% BSA. To assay the migratory behavior of cells, the CytoSelect Cell Haptotaxis Assay Kit (CellBiolabs, CA) was used. This kit contains inserts with polycarbonate membranes having pores of size of 8μm. Collagen I is coated on the bottom side. 500μl of medium containing 10% FBS was placed in the well, while 300 μl of the cell suspension was loaded in the inserts. The cells were incubated at 37°C and were allowed to migrate for 4 hours through the pores in response to Collagen I and FBS. The medium was aspirated from the inserts, and the top of the polycarbonate membrane was cleaned using cotton swabs to remove non-migratory cells. The inserts were then transferred to clean wells containing 300μl of Lysis Buffer/CyQuantR GR dye solution. After 10 minute incubation at room temperature and transfer, the florescence of the CyQuant® GR dye solution was measured at 480nm/520nm in a plate reader. The measured fluorescence is directly proportional to the number of cells that had migrated to the bottom side of the polycarbonate membrane.

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