Cells in 250μl 0.4% soft agarose (Life Technologies, Carlsbad) mixed with 10% FBS containing medium were plated at 2,000/well on top of a 500μl solidified underlayer of 0.8% agarose in the same medium in a 24-well tissue culture plate. Doxycycline (1μM) or equivalent control vehicle, DMSO, were administrated after 5 days when small colonies formed. After 2–3 weeks of incubation, cell colonies were visualized by overnight staining with 1 ml p-iodonitrotetrazolium violet staining (Sigma, St Louis, MO, USA). Then scan stained soft agarose plates with a scanner and colony number was quantified using Image J.
Soft agarose
Manufactured by Thermo Fisher Scientific
0.4% soft agarose is a laboratory-grade agarose solution used in various gel electrophoresis applications. It provides a soft, porous matrix for the separation and analysis of macromolecules, such as DNA, RNA, and proteins.
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2 protocols using soft agarose
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Colony Formation Assay
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Colony Formation Assay
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Cells in 250μl 0.4% soft agarose (Life Technologies, Carlsbad) mixed with 10% FBS containing medium were plated at 2,000/well on top of a 500μl solidified underlayer of 0.8% agarose in the same medium in a 24-well tissue culture plate. Doxycycline (1μM) or equivalent control vehicle, DMSO, were administrated after 5 days when small colonies formed. After 2–3 weeks of incubation, cell colonies were visualized by overnight staining with 1 ml p-iodonitrotetrazolium violet staining (Sigma, St Louis, MO, USA). Then scan stained soft agarose plates with a scanner and colony number was quantified using Image J.
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