Migration Assay Protocol:
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Materials to be prepared beforehand:
1) FBS free medium
2) 10% FBS medium
3) Cell migration filter insert ( Transwell®, 12mm Diameter, 12 μm Pore Size.)
Procedures:
1) Coat the underside of filter inserts with 20μg/ml of fibronectin overnight at 4 oC Air dry the insert before the cells were added.
2) Trypsinize cells and wash twice with FBS free medium.
3) Count cells to 4×105/ml.
4) Add 0.5ml cells in FBS free medium 10 the top of insert .
5) Add 1ml of medium (with 10% FBS and 10μg/ml Collagen I ) to the lower chamber .
6) Put filter insert into the lower chamber and incubate overnight at 37 oC.
7) Remove cells on the topside of the filter by scrubbing twice with cotton tipped swab moistened with no FBS medium.
8) Stain cells with Diff-QuikTM stain kit.
9) Wash with distilled water.
10) Mount.
