DNA Staining for Flow Cytometry

1. Collect about 2 x 10e6 cells for each sample. Spin at 1200 rpm for 3 minutes. Suck out medium without disturbing the pellet (the cells). Wash once with

    PBS.
2. Resuspend cells thoroughly in 50 µl of PBS. You must not add alcohol to a cell pellet or else the cells will clump together permanently. Add 1 ml of

    ice-cold 80% ethanol by pipetting it forcibly into the cell suspension. Alternatively, add the ethanol slowly while vortexing the mixture at low speed. Mix

    thoroughly or else the cells will clump permanently. Fix for 30 minutes to overnight at 4°C. If you need to store the cells longer, put them at -20°C for

    as long as several weeks.
3. Resuspend the cells and transfer to a microfuge tube. Spin at 6000 rpm for 2 minutes. Suck out ethanol carefully, without disturbing the pellet. The

    cells don’t stick to the bottom of the tube when in alcohol. Resuspend the cells in 500 µl of 2 mg/ml RNase A in PBS. Incubate for 5 minutes at room  

    temperature.
4. Add 500 µl of 0.1 mg/ml Propidium iodide, 0.6% NP-40 in PBS. Mix thoroughly. Incubate for 30 minutes at room temperature, protected from light.
5. Spin at 6000 rpm for 2 minutes. Suck out liquid without disturbing the pellet. Resuspend the cells in 500 µl of PBS. Store at 4°C until samples are

    needed.

6. Pipette the cell suspension into FACS tubes. Pipette it onto the filter on the FACS tube at an angle. The sample is now ready for flow cytometry.

 

 

 

 

​Click here to download the protocol

By: Andrew Morrison

Mailing Address

Arthur and Sonia Labatt Brain Tumour Research Centre

The Hospital for Sick Children

Peter Gilgan Centre for Research and Learning

686 Bay Street, 17-9702
Toronto, ON  M5G 0A4

Canada