Five hundred microliter of 0.8% low-melting agarose (Life Technologies, Carlsbad, CA) in RPMI containing 10% FBS was added in a 24-well tissue culture plate as underlayer. Five thousand cells in the culture medium were mixed with 0.8% soft agarose in equal volume ratio and were plated on the top of the underlayer drop by drop. After 12-14 days of incubation, cell colonies were visualized by overnight staining with 250 μl p-iodonitrotetrazolium violet staining (Sigma, St Louis, MO). The plates were scanned with a scanner and colony number in each well was quantified using Image J.
P iodonitrotetrazolium violet staining
Manufactured by Merck Group
Sourced in United States
P-iodonitrotetrazolium violet staining is a colorimetric assay used to detect the presence of viable cells. It functions by measuring the reduction of the tetrazolium dye, which changes color when reduced by cellular enzymes, thereby indicating the metabolic activity of cells.
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3 protocols using p iodonitrotetrazolium violet staining
1
Soft Agar Colony Formation Assay
2
Colony Formation Assay
3
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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