Detection of cell cycle subcompartments by flow cytometric estimation of DNA–RNA content in combination with dual-color immunofluorescence
✍ Scribed by Ingrid Schmid; Steve W. Cole; Yael D. Korin; Jerome A. Zack; Janis V. Giorgi
- Publisher
- John Wiley and Sons
- Year
- 2000
- Tongue
- English
- Weight
- 323 KB
- Volume
- 39
- Category
- Article
- ISSN
- 0196-4763
No coin nor oath required. For personal study only.
✦ Synopsis
Background: Correlated flow cytometric measurements of phenotype and DNA-RNA content offer detailed information on cell cycle status of subpopulations in heterogeneous cell preparations in response to stimulation. We have developed a method for flow cytometric analysis of DNA-RNA content that has been optimized for simultaneous measurement of dual-color immunofluorescence. Methods: Nucleic acid staining was performed at low pH in the presence of saponin. DNA was stained with 7-aminoactinomycin D (7-AAD) and RNA with pyronin Y(G) (PY); both dyes were used at low concentrations, and 7-AAD was exchanged with nonfluorescent actinomycin D after DNA staining to minimize fluorochrome-fluorochrome interactions. For cell surface antigen staining, allophycocyanin was combined with pH-independent Al-exa™488 instead of fluorescein-isothiocyanate (FITC) because FITC is pH sensitive.
Results: This method identified cell cycle subcompartments in CEM cells comparable to published results on cell lines using other dyes and staining methods. Measurement of DNA-RNA content in CD8 lymphocyte subsets of human peripheral blood mononuclear cells costimulated with CD3/CD28.2 showed that, after 48 h of stimulation, 80% of CD8 ϩ T cells were in the proliferative state, whereas 86% of CD8 ϩ non-T cells remained in G 0 . Conclusions: This technique permits the clear identification of cellular subpopulations by phenotype and assessment of their cell cycle status.