Cultrex® BME Cell Invasion Assay

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	In the lower chnaber add medium supplemented with 10% FBS. Incubate plates at 37 °C (5% v/v CO2) for 24 h,	Read plate at 485 nm excitation, 520 nm emission

Protocol tips
In the lower chnaber add medium supplemented with 10% FBS. Incubate plates at 37 °C (5% v/v CO2) for 24 h,
Downstream tips
Read plate at 485 nm excitation, 520 nm emission

Publication protocol

Cell migration through basement membrane extract was assayed using Cultrex basement membrane extract cell invasion assay in a 24- or 96-well plate format (Trevigen Inc., Gaithersburg, MD). Cells were starved overnight in serum-free medium, detached by treatment with Accutase, and resuspended in serum-free medium to give 106 live cells ml−1. In the final assay setup, each upper well of the modified Boyden chamber contained 5 × 104 cells (24-well format) or 0.5 × 104 cells (96-well format). To test the effects of concanamycin, the inhibitor was added to an aliquot of the cell suspension to give a final concentration of 1 μm in the upper chamber (0.5% v/v DMSO, also included in control wells). Soluble recombinant receptor activator of nuclear factor κB ligand (PeproTech, London, UK) was used at 10 μg ml−1. As chemoattractant, the bottom well of each chamber contained medium supplemented with 10% FBS. Plates were incubated at 37 °C (5% v/v CO2) for 24 h, after which time any cells that had passed through the basement membrane extract/polycarbonate membrane were detached. Cells were quantified by incubating with calcein-AM and measuring the internalized fluorescence signal in a plate reader accessory attached to a Cary Eclipse scanning fluorimeter or FlexStation 3 (excitation at 485 nm and emission at 520 nm).

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