Propidium iodide (PI) is an ethidium bromide analog that emits red fluorescence upon intercalation with double-stranded DNA. PI does not permeate viable cell membranes, but passes through disturbed cell membranes and stains the nuclei. PI is often used in combination with a fluorescein compound, such as Calcein-AM or FDA, for simultaneous staining of viable and dead cells. The excitation and emission wavelengths of PI-DNA complex are 535 nm and 615 nm, respectively.
1.Prepare 10-50 μM PI solution with PBS or an appropriate buffer.a)
2.Add PI solution with 1/10 of the volume of cell culture medium to the cell culture.b)
3.Incubate the cell at 37oC for 10-20 min.
4.Wash cells twice with PBS or an appropriate buffer.
5.Observe the cells under a fluorescence microscope with 535 nm excitation and 615 nm emission filters.
a) Since PI may be carcinogenic, extreme care is necessary during handling.
b) Or you may replace the culture medium with 1/10 concentration of PI buffer solution.
1. I. W. Taylor, et al., An Evaluation of DNA Fluochromes, Staining Techniques, and Analysis for Flow Cytometry. I. Unperturebed Cellpopulations. J Histochem Cytochem. 1980;28:1224-1232.
2. W. M. J. Vuist, et al., Potentiation by Interleukin 2 of Burkitt’s Lymphoma Therapy with Anti-Pan B (Anti-CD19) Monoclonal Antibodies in a Mouse Xenotransplantation Model. Cancer Res. 1989;49:3783-3788.
3. A. K. El-Naggar, et al., Single- and Double-stranded RNA Measurements by Flow Cytometry in Solid Neoplasms. Cytometry. 1991;12:330-335.
4. C. Souchier, et al., Methods for Cell Proliferation Analysis by Fluorescent Image Cytometry. Cytometry. 1995;20:203-209.
5. T. Irino, et al., Establishment of Real-Time Polymerase Chain Reaction Method for Quantitative Analysis of Asparagine Synthetase Expression. J Mol Diagn. 2004;6:217-224.
Fig. 2 Cell staining with PI