CytoSelect™ 24-Well Cell Migration and Invasion Assay Combo Kit, 8 µm

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Add 300 µL of the cell suspension solution to the inside of each insert. Incubate for 22 hours in a cell culture incubator.	Measure the OD 560nm in a plate reader

Protocol tips
Add 300 µL of the cell suspension solution to the inside of each insert. Incubate for 22 hours in a cell culture incubator.
Downstream tips
Measure the OD 560nm in a plate reader

Publication protocol

Migration and invasion experiments were conducted separately using a CytoSelect™ 24-Well Cell Migration and Invasion Assay (8 μm, Colorimetric Format) (Cell Biolabs, Inc., San Diego, CA, USA) and 24-well migration and invasion plates containing polycarbonate membrane inserts with 8 μm pores. The upper membrane surfaces of the inserts for the invasion assay were covered in a uniform layer of dried basement membrane matrix solution. Melanoma cells transfected with hsa-miR-4286 anti-miRTM miRNA inhibitor (Cat. № AM17000, Ambion, Life Technologies, Carlsbad, CA, USA) or anti-miRTM negative control #1 (Cat. № AM17010, Ambion, Life Technologies, Carlsbad, CA, USA) were detached with 0.25% trypsin in HBSS (Gibco®, Paisley, UK) after 24 h of transfection, washed with PBS and suspended in FBS medium RPMI-1640 free to final concentration of 1 × 105 cells per ml. The cell suspension was then placed inside each insert and the bottom well of the migration plate was filled with the medium containing 10% FBS. After 22 h of incubation at 37°C in 5% CO2, the cells on the interior of the inserts were mechanically removed, and the migrated cells were fixed and stained with the kit’s Cell Stain Solution and dried according to manufacturer’s instructions. The stained cells were analyzed on a Floid® Cell Imaging Station (Life Technologies, Bothell, WA, USA) in three random fields. The cells were dissolved using the kit’s Extraction Solution for 10 min and transferred to 96-well plates to evaluate the optical density at 560 nm using EFOS-9305 spectrophotometer (Shvabe-photosystems, Moscow, Russia). The relative migration/invasion activity was determined by calculating the ratio of the average absorbance in hsa-miR-4286 anti-miRTM miRNA inhibitor cells to the average absorbance of anti-miRTM negative control #1 cells. The experiments were performed in triplicate

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