Propidium iodide (PI) is a red-fluorescent nuclear and chromosome counterstain which can bind to double stranded DNA by intercalating between base pairs.
Propidium iodide (PI) is a red-fluorescent nuclear and chromosome counterstain which can bind to double stranded DNA by intercalating between base pairs. It is a cell impermeant dye, which can be excluded from viable cells. Thus it is widely used for the evaluation of apoptosis in different experimental models. Propidium iodide is excited at 488 nm and, with a relatively large Stokes shift, emits at a maximum wavelength of 617 nm. General protocol:
1. Trypsinize and harvest cells in the appropriate manner and wash in PBS.
2. Fix cells into 0.5 ml 70% EtOH for at least 1h. 70% EtOH should be pre-cooled to -20 °C overnight.
PAUSE POINT: Cells can be stored in ethanol solution at –20°C for several weeks.
3. Spin down cells for 2 min at 2,000 rpm.
4. Discard the supernatant. Be careful to avoid cell loss.
5. Wash in PBS for two times as: resuspend cell pellet in 0.5mL 1xPBS, spin down cells for 2 min at 2,000 rpm, discard the supernatant and repeat these steps.
6. Resuspend cell pellet in 0.5 ml PBS containing 10 μg/ml RNase A and 20 μg/ml PI stock solution, transfer to FACS tubes.
Note: The addition of RNase A could ensure that only DNA is stained.
7. Incubate at room temperature in the dark for 30 min.
8. Analyze cells by flow cytometry. Use 488-nm laser line for excitation. Measure red fluorescence (>600 nm) and side scatter.
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