Oris™ Pro Cell Migration Assay

Protocol tips

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	- After washing step, cells are incubated for 18 hours and later cells were stained with Calcein AM (0.5 µg/ml)

Protocol tips
- After washing step, cells are incubated for 18 hours and later cells were stained with Calcein AM (0.5 µg/ml)

Publication protocol

The effect of the SP2024 peptide on cells migration was investigated using two different assays; a real time migration assay system based on electrical impedance (RT-CIM, ACEA Biosciences, CA) and a wound healing type assay (Oris Pro Migration assay, Platypus Technologies, CMA 1.101). The RT-CIM assay uses a CIM 16-well plate (Roche, Basel, Switzerland) composed of a top and bottom chamber separated by a microporous polycarbonate membrane (8 µm). The membrane was coated with fibronectin (20 µg/ml) and 45,000 cells/well (HUVEC or LEC) in serum free media, with or without peptide was added to the top compartment. Media with chemoattractant (i.e., fully supplemented EBM-2 or EBM-2MV) was added to the bottom compartment of the chamber, and the plate was incubated at 37°C for 20 hours. The sensors integrated on the bottom side of the membrane monitored and continuously recorded changes in impedance as the cells moved through the membrane. Breast cancer cells MDA-MB-231 are not suitable for RT-CIM type experiments due to their thin elongated phenotype that results in low signal, thus the inhibition of migration was investigated using a wound healing type assay. This assay was performed using the Oris Pro Migration assay (Platypus Technologies, CMA 1.101). Briefly, 25,000 cells/well in fully supplemented media were added to the 96-well plate containing stoppers that were used to block the migration of the cells to the center region of the wells. Cells were allowed to adhere for 4 hours, after which the stoppers were removed. After 18 hours cells were stained with Calcein AM (0.5 µg/ml) (Invitrogen, CA) and the cells that migrated to the center of the well were quantified by fluorescence intensity measured with a Victor V plate reader.

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