SPLInsert™ Hanging

Protocol tips

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	Incubate for 24 hours and then add serum free media

Protocol tips
Incubate for 24 hours and then add serum free media

Publication protocol

For single culture assays, HaCaT cells or fibroblasts (7 × 104 cells per well) were seeded in the insert of a 12-well invasion assay kit (SPL), and the bottom portion was filled with media. For co-culture assays, one cell type was seeded in the insert, and the other type was seeded in the bottom, using equal numbers of each (for example, HaCaT cells in the insert and fibroblasts in the bottom and vice versa). The membrane allows the exchange of media during incubation. After 24 h, serum-free media was added to the insert to allow for chemotaxis, and cells in both the insert and bottom were treated with either DMSO control or G:T=1:1 for 48 h. Cells on the upper side of the insert were then removed using a cotton swab, and cells on the lower side of the insert were fixed with 4% formaldehyde. After washing with PBS, invaded cells were stained with a 1% crystal violet solution, and photographs were acquired at × 4 magnification using an Olympus IX70 microscope. Invasive ability was measured using the Image J program

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Manufacturer protocol

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