Hanging drop aggregation assay

Description
This assay is used for the aggregation property of cancer cells. It is a very critical parameter for measurement of cell metastasis. Factors that critically affect tumor cell metastasis are aggregation and adhesive properties of the cells. This property is usually altered in the tumor cells.

Procedure
1.Trypsinize the cells in the presence of EDTA, wash twice in PBS, and resuspend at 2.5 x105 cells/ml in the appropriate medium containing 10% FBS.
2.Pipette drops (20μl each) of medium, containing 5000 cells/drop, onto the inner surface of the lid of a Petri dish.
3.Then place the lid on the Petri dish so that the drops will be hanging from the lid with the cells suspended within them.
4.To eliminate evaporation, place 8 ml of serum-free culture medium in the bottom of the Petri dish.
5.Incubate cells overnight at 37°C.
6.Invert the lid of the Petri dish and take the photograph using inverted tissue culture microscope at X40 magnification.