PI determination of cellular DNA content
**These protocols are meant to be modified with your experiment specifics in mind. This can be done in conjuunction with the RCF staff if you require any assistance
The protocol is in part based on: Determining Cell Cycle Stages by Flow Cytometry, Current Protocols in Cell Biology
Seed, Culture, Synchronize and Fix cells
Seeding cell density will be dependent on timeframe of any treatments, cell type, culture dish/flask size, etc. A typical 48h culture in a 6-well plate would use approximately 1-2x105 cells per 6-well plate. Appropriate controls will also be experiment-specific.
The cells should be seeded and then synchronized (using serum starvation for 12-72 hours depending on the cell type). Serum starvation does not work well for cells with slow doubling times. Other alternatives include nocodazole and a good reference is http://immuneweb.xxmu.edu.cn/download/CellCycleMethods.pdf
At the endpoint you typically want cells to be largely confluent without being over-confluent, as apoptosis may occur and/or result in inhibition of mitosis, which will significantly affect your results.
Remove the supernatant and any detached cells via aspiration and wash the adherent cells with PBS. Trypsinize the adherent cells, collect them, and stop the trypsin with neutralizing solution if applicable.
Pellet the cells (this is experiment-specific, but a general guideline is 500xg for 5 minutes) and aspirate the supernatant. Resuspend the pellet at 2 x 106 cells per mL and make sure you have a single cell suspension via vigorous pipetting.
In an appropriate tube for flow cytometry, add 4.5mL of cold 70% methanol/ethanol and add the cells dropwise to the alcohol while gently vortexing to ensure the cells remain single-cell suspensions and do not clump together.
Fix on ice for 30 mins.
Staining with Propidium Iodide
Pellet cells (using the same speed as above; here I use 500xg for 5 minutes) via centrifugation and discard the supernatant. Be certain to remove as much ethanol as possible.
Wash with 5mL PBS twice to remove all alcohol.
Resuspend the cell pellet in 1mL PI solution (20g/mL PI, 0.1% Triton X-100 and 50L RNAse in PBS as solvent).
The RNAse amount will depend on the RNAse provider that you use and here is just a guideline. The Triton will permeabilize the cell and the RNAse is required in order to eliminate dsRNA, which will bind PI and give a large confounding background fluorescent signal. You want a fluorescent signal to be dependent only on the DNA content.
Incubate at 37oC for 15 min. Dilute as necessary with an isotonic buffer (i.e. PBS, sheath fluid, etc.) and proceed to flow cytometric measurement of the PI fluorescence.
***For flow cytometry it is crucial to keep the events per second below 500 if possible and to perform singlet gating to exclude coincident events (FSCA vs. FSC-H or FSC-W followed by the same using SSC). It is also common to threshold on PI positive fluorescence (FL-2/B3/etc.) to eliminate debris or unlabeled cells.
Addendum: determination of DNA index
The procedure outlined above would be followed in order to stain the DNA complement with PI (this gives the best CV). Next we need the MFI of the Go/G1 peak in the control and experimental population to calculate the DNA Index (DI).
DI = MFIexperimental/MFIconrol
A general guideline is that if the DI<0.95 of >1.05 you have a good case for aneuploidy, provided that the CV of your Go/G1 peak is ~3%. Above that, your statistical variability increases and the requisite difference between the MFIexperimental/MFIconrol needed to say you are observing aneuploidy also increases.