2 dimensional determination of cellular DNA content

EdU kits are generally easier and more sensitive than the older BrdU protocols. The protocol for EdU kits is supplied by the manufacturer, but if you prefer to use BrdU a common protocol is listed below.

Seed, Synchronize and Grow cells with BrdU

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

Incubate cells with ~10mM BrdU for an appropriate time (cell type and experiment dependen). This will be ~1 hour but highly variable. Base the timepoint on a pilot experiment, publications or knowledge of your cell doubling time.

BrdU is photo-labile and you MUST keep the samples protected from light as much as possible during all subsequent steps.

Harvest, Fix and Label the BrdU

Wash the cells 3x 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 (or longer if you are batch processing).

Pellet the cells (experiment-dependent here I use 500xg for 5 minutes) noting that the cells will be “lighter” after alcohol fixation.

Resuspend the pellet in fresh 2M HCl (diluted in ddH20 from the concentrated stock) and incubate for 30 mins at room temperature, mixing by inversion every 10 mins. This is necessary to make the incorporated BrdU accessible to the antibody used for detection.

Wash twice with PBS to remove any HCl and resuspend in 100L your favorite flow cytometry staining buffer (PBS with 2%BSA, etc.) containing 0.2% TWEEN/TRITON X-100.

Stain using anti-BrdU antibody (eg APC anti-BrdU from BioLegend cat# 339807) following the manufacturer’s recommendations (typically 1g per 106 cells for 15-20 mins)in the dark followed by 2g of PI.

Since we are doing intracellular (nuclear) staining, I recommend washing 3x with your staining buffer (containing detergent) to remove un-bound antibody.

Run the samples on a flow cytometer remembering to do singlet gating and PI thresholding if desired. You will want to look at the PI (X-axis) vs. BrDU (Y-axis) graph to determine the Go/G1, S and G2/M populations.