DNA content may be measured in association with other nuclear or cellular components such as cyclins by dual fluorescence analysis in which PI/DAPI is used to stain the DNA and for example FITC or APC conjugated antibody is used to define the antigen of interest. When using DAPI most antibody fluorophores can be used except for Pacific Blue. Whilst with PI which emits at 619nm, PE, PerCP or PE-Cy5 or conjugated antibodies cannot be used as as PI emits at the same wavelength as these fluorophores.
Surface immunophenotyping can also be used in conjunction with DNA content using PI or DAPI. This can also be combined with intracellular staining, for example keratinocytes were surface labelled with anti-CD34-APC and CD49-PE; then fixed with 2% PFA for 15 min at 4C, then permeabilised with 0.1% saponin for 5 minutes at RT. After washing cells were then labelled intracellularly with anti-B-Catenin-FITC and then stained with 1ug/ml DAPI for cell cycle analysis. All populations of cells were then analyzed for DNA content and the percentage of proliferating cells determined for each population, see figure.
Cell death associated cell cycle antigens for example p53 and Bax in reagard to apoptosis and p16 can also be measured to indicate the level of senescence, see figures.
List of protocols .
- 3-D histogram plot
- PI cell cycle analysis with intracellular antigen protocol
- 7-AAD cell cycle analysis and intracellular antigen staining
- DAPI cell cycle analysis with intracellular antigen staining protocol
- PI cell cycle analysis with surface staining protocol
- DAPI cell cycle analysis with surface staining protocol