The simultaneous staining of DNA with propidium iodide (PI) and nuclear protein with fluorescein isothiocyanate (FITC) provides information analogous to the acridine orange (AO) staining of partially denatured DNA in whole cells. In the procedure described, nonionic detergent is used to strip the nuclei of cytoplasm and the nuclei are then stained without washing. The technique is rapid and allows for the discrimination of multiple cell cycle compartments. The analysis is readily applied to solid tumors. Propidium iodide fluorescence of stained nuclei is proportional to the DNA content under most conditions. The addition of FITC staining provides a degree of separation of overlapping cell populations and cell cycle compartments. The preparation of nuclei that are free of cytoplasmic tags is important for the accurate measurement of both DNA and nuclear protein. Because PI stains double-stranded nucleic acid, including RNA, the presence of cytoplasmic tags will cause an increase in the coefficient of variation (CV).
ASJC Scopus subject areas
- Cell Biology