Comparison of Vindelov et al. and bromodeoxyuridine/DNA double-staining flow cytometry methods for analysis of cell cycle distribution in rat thymocytes.
This study compares the cell cycle distribution in rat thymocytes obtained by means of bromodeoxyuridine (BrdUrd) labeling of S-phase cells and the analysis of the S-phase fraction obtained according to the technique of Vindelov et al. (Cytometry 3:332-338, 1983). The proportion of BrdUrd-labeled cells was analyzed in single cell suspensions of adult rat thymocytes after in vivo injection of BrdUrd and the results then compared with those obtained after measuring the cell DNA contents according to the Vindelov et al. method. The percentage of BrdUrd-positive cells was greater than the S-phase fraction obtained using the Vindelov et al. technique. By contrast, no major differences were observed between the percentage of BrdUrd-positive cells and the S-phase fraction obtained after analyzing the DNA histograms of the same data files with the RFIT mathematical model. The elimination of trypsin treatment used in the Vindelov et al. method did not alter the results, whereas the use of DNA denaturation with 2N HCl was shown to increase the percentage of S-phase rat thymocytes (calculated from DNA histograms) independently of whether trypsin treatment was used or not. However, the value of the S-phase fraction was not as great as that obtained after BrdUrd labeling. Thus when comparing BrdUrd-labeling and the Vindelov et al. technique, important differences in the percentage of S-phase adult rat thymocytes were observed. Selective G0/G1 cell loss during washing and centrifugation steps performed after the DNA denaturation used for BrdUrd detection was the main reason for these differences.
Orfao
A
A
Unit of Cytometry, University of Salamanca, Spain.
Carbajo
S
S
Dolbeare
F
F
Ciudad
J
J
Lopez
A
A
Carbajo-Perez
E
E
D000818:Animals; D001973:Bromodeoxyuridine; D004247:DNA / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D016193:G1 Phase; D008297:Male; D009691:Nucleic Acid Denaturation; D051381:Rats; D017207:Rats, Sprague-Dawley; D016192:Resting Phase, Cell Cycle; D016196:S Phase; D013535:Suspensions; D013950:Thymus Gland / Q000166:cytology* / Q000187:drug effects; D014357:Trypsin / Q000494:pharmacology
D005456:Fluorescent Dyes; D013535:Suspensions; D004247:DNA; D014357:Trypsin; D001973:Bromodeoxyuridine