Multiparametric cell-cycle analysis of peripheral blood-activated lymphocyte subsets using staining based on the TdT method for incorporated BrdUrd
✍ Scribed by F. Dolbeare; N. Lopez; A. Lopez; M. L. Amigo; J. Ciudad; M. C. Del Cañizo; J. F. San Miguel; A. Orfao
- Publisher
- John Wiley and Sons
- Year
- 1996
- Tongue
- English
- Weight
- 638 KB
- Volume
- 25
- Category
- Article
- ISSN
- 0196-4763
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✦ Synopsis
This study describes a new method for the shultaneous assessment of the distribution of a cell population in the GO/Gl, s, and G 2 N cell-cycle phases by using multiparameter flow cytometry and single staining based on BrdUrd incorporation.
Both the K562 cell line and PHA-stimulated peripheral blood lymphocytes (PBL) were analyzed. Cells were cultured in the presence of BrdUrd for 30 min prior to cell harvesting. Once collected, cells were exposed to ultraviolet light for 5 min and then fixed immediately in 70% ethanol (-20°C) for at least 30 min. Once fixed, the cells were placed for 30 min at 37°C in the presence of terminal deoxynucleotidyl transferase (TdT) and dUTP labeled with digoxigenin; they were then stained with FITC-labeled anti-digoxigenln.
Our results show that GO/Gl, S, and G 2 N cell populations can be clearly discriminated according to FITC fluorescence and light-scatter parameters. In this way, S-phase cells can be identified by their FITC staidng. From the cells which were negative for anticould be resolved in a forward scatter, side scatter, and fluorescence pulse-width three-dimensional plot; the values obtained for GO/Gl cells were lower than those obtained for G2/M cells in all three parameters. Multiparameter analysis of PBL stained for two surEdce antigens (CD3 and CD8) and for BrdUrd by direct or indirect TdT method permitted cell-cycle analysis of Merent subpopulations, including CD3 + /CDS + , CD3 + /CD8-, CD3-/CDS + , and CD3-/CD8-.
6 1996 wiley-~iss, hc.