Soft Agar Colony Formation Assay

As described previously^{6 (link)}, a bottom agarose layer (1%) was laid in 12-or 6-well plates. Cells (1 × 10⁴ cells/well), resuspended in 2 mL of 0.5% agarose solution in complete medium, were overlaid and incubated for 25 days. Colonies formed were stained using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma), and those greater than 80 μm in diameter were counted using the GelCount system (Oxford Optronix, UK) according to the manufacturer’s instructions.

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Gagliardi M., Pitner M.K., Park J., Xie X., Saso H., Larson R.A., Sammons R.M., Chen H., Wei C., Masuda H., Chauhan G., Kondo K., Tripathy D., Ueno N.T., Dalby K.N., Debeb B.G, & Bartholomeusz C. (2020). Differential functions of ERK1 and ERK2 in lung metastasis processes in triple-negative breast cancer. Scientific Reports, 10, 8537.

Publication 2020

MTT GelCount system

Agarose Bromide Cells

Corresponding Organization :

Other organizations : The University of Texas MD Anderson Cancer Center, The University of Texas at Austin, Austin College

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Variable analysis

independent variables

Cell density (1 × 10^4 cells/well)

dependent variables

Number of colonies greater than 80 μm in diameter

control variables

Bottom agarose layer (1%)
Agarose solution concentration (0.5%)
Incubation time (25 days)

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