DNA fragmentation was assayed using a Cell Death Detection ELISA PLUS (Sigma Aldrich, St. Louis, MO). Anchorage-independent growth (AIG) was assayed as described previously (9 (link)). For each cell line, 1 × 104 cells were suspended in a top layer of 0.36% noble agar (USB Corporation) and a bottom layer of 0.4% agar both in RPMI-1640 + 10% FBS in a 6-well culture dish. After 1–3 weeks, colonies were stained with Nitrotetrazolium Blue chloride (NBT; Sigma) and photographed with a S600 Canon camera. Colonies were quantified with ImageJ analysis software.
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Quantifying Anchorage-Independent Growth
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Corresponding Organization :
Other organizations : University of Colorado Anschutz Medical Campus, University of Colorado Denver
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Variable analysis
independent variables
- Cell lines
- DNA fragmentation
- Anchorage-independent growth (AIG)
- Number of cells seeded (1 × 10^4)
- Agar concentration (0.36% in top layer, 0.4% in bottom layer)
- Culture medium (RPMI-1640 + 10% FBS)
- Culture dish (6-well)
- Staining method (Nitrotetrazolium Blue chloride)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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