Propidium Iodide Staining for DNA Ploidy

 

Solution:

 

  1. Propidium Iodide (w/o Ca2+ and Mg2+)

Stock10 mg/ml in Ethanol

 

  1. 70% Ethanol in 1X PBS (w/o Ca2+ and Mg2+)

 

Note: Be sure to use molecular grade 200-proof Ethanol.

DO not use Ethanol from metal can.

 

Procedure:

 

  1. Start with 1X106cells/mL.

Tumor or Lymphocytes (used for control for 2N population)

 

  1. Harvest cells (by trypsinizing of need be). Obtain pellet.

 

  1. Wash with 1XPBS ( Resuspend and spin )

 

  1. Resuspend with cold 70% Ethanol drop by drop

 

  1. Store @ 4C for minimum of 24 hour/overnight.

 

  1. Spin down

 

  1. Add RNase A to final concentration 100 units/ml.

Dissolve RNase A (10mg/ml) in 1 ml of PBS

Add 250μl in each tube

 

  1. Incubate for 30 min. at room temperature.

 

  1. Spin down

 

  1. Remove supernatant and add 500ul of 1XPBS

 

  1. Spin down

 

  1. Resuspend pellet with 1ml of 1XPBS

 

  1. Add Propidium Iodide to final concentration of 20μg/ml

2μl of PI (stock; 10mg/ml) in 1 ml

 

  1. Protect samples from light and store at 4C until FACS analysis