The trans-well invasion assays were performed as described previously (9 ). Briefly, cells were incubated overnight in the culture media with 0.2 % FBS. The next day, the cells were trypsinized, re-suspended in the low serum media, and seeded at 5 × 104 for MDA or 1 × 105 for CN34 per well into the growth factor reduced matrigel invasion chambers (8 µm pore size, BD Biosciences). After 18–22 hr, the chambers were washed with PBS twice and the cells on the apical side of each inserts were scraped off. The invaded cells fixed with 4% paraformaldehyde were stained with DAPI using VectaShield (Vector Laboratories) and were counted in four fields per insert and then quantified with ImageJ (National Institutes of Health).
Growth factor reduced matrigel invasion chambers
Manufactured by BD
Growth factor reduced Matrigel invasion chambers are a cell culture tool used to evaluate the migratory and invasive potential of cells. The chambers consist of a porous membrane coated with a reduced concentration of basement membrane components, which cells must degrade and migrate through to reach the lower chamber.
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4 protocols using growth factor reduced matrigel invasion chambers
1
Quantitative Trans-well Invasion Assay
2
Transwell Invasion Assay with Endothelial Cells and Macrophages
3
Soft Agar and Invasion Assays
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For soft agar assay, 6-well tissue culture plates were coated with 0.5% agar (Biozym) in DMEM. Cells to be assayed were resuspended at 37°C in 0.35% agar in DMEM. After solidification of agar, full medium was added on the top. 5000 cells were seeded per well in triplicates. After two weeks plates were stained with crystal violet and colonies were counted in 10 optical fields/well. For the invasion assay, growth factor reduced matrigel invasion chambers with a pore diameter of 8 μM (BD Biosciences) were used.
4
Matrigel Invasion Assay Protocol
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Cells were suspended in serum-free RPMI or DMEM media containing 0.1% BSA and added to 8.0 μm pore Growth Factor-Reduced Matrigel Invasion Chambers (BD Biosciences) at 105 cells/μL in four wells per experimental condition. NIH-3T3 conditioned media was used in the lower chamber of the invasion plate as a chemoattractant. After 24 hours, each well was washed with PBS, and cells on top of the membrane were removed with cotton-tipped applicators (Puritan Medical Products, Guilford, ME). Invaded cells were stained by submerging each well in a solution of 0.5% crystal violet, 20% methanol for 5 minutes. Nine fields per well were imaged at 100X magnification. The invasive cells in each field were counted using ImageJ software. Normalized invasion was calculated as a percentage compared to a control condition.
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