Cyan analyzer

CT26 cells were seeded in a 6-well plate at 3 × 10⁵ cells per well, incubated with increasing concentrations of RB (0, 100, 200, and 300 μM) for 24 h, and processed for cell cycle analysis. For cell death evaluation, both murine and human colon cancer cells were treated with RB for 1, 4, 8, and 16 h, after which both floating and attached cells were collected for further analysis.
Secondary to spectral overlap of RB with propidium iodide, DAPI was used to stain DNA and label dead cells. For cell cycle analysis, harvested cells were fixed with 80% ethanol on ice for 30 min and then incubated with 1 ml of DNA staining solution (1 μg/ml DAPI, 0.1% NP-40 in PBS) at room temperature for 30 min. DNA content was determined with FACS on a CyAn analyzer (Beckman Coulter, Brea, CA, USA) and analyzed with the FlowJo software (Ashland, OR, USA).
Cell death was determined with an Annexin V-Biotin Apoptosis Detection Kit. Briefly, cells were first stained with biotin-conjugated Annexin V in 1x binding buffer at room temperature for 15 min and then incubated with streptavidin-APC for 30 min. After being washed with PBS, cells were resuspended in PBS containing DAPI (0.2 μg/ml) for 5 min followed by FACS analysis. Cell death profile was analyzed with the FlowJo software. Necrotic cells were defined as DAPI+ and DAPI+/Annexin V+, and apoptotic cells were defined as Annexin V+ only.^{46 (link)}