• Three-color immunofluorescence;
  • cell cycle;
  • T-cell differentiation;
  • T-cell receptor;
  • in vivo cell tracing


We present a comparison of two different methods for simultaneous detection of bromodeoxyuridine and cell surface markers. Both methods use enzymatic generation of single-etrand DNA with nuclease. The biological system used is the murine thymus, in which in vivo DNA synthetizing cells were labeled by injection of BrdUrd and analyzed at different time points after the nucleoside pulse. The surface proteins detected were CD4 and CD8 differentiation markers and the T-Tell receptor. Extraction of DNA-associated proteins with 0.1N HC1 and detergent is necessary for the action of EcoR1 and Exonuclease III, but this treatment destroys phycocyanins and induces cell aggregation, as shown using the doubletdiscrimination module. For DNAse I action, cells could be treated with paraformaldehyde and a low concentration of Tween 20, and this treatment was adequate for surface staining preservation (even writh phycocyanins) and BrdUrd detection. Both methods were adequate for cell cycle studies, but only 7-aminoactinomycin D could be used as total DNA dye after DNAse action, and good results needed long (48–72 h) incubation in the fixative-eetergent mixture. The DNAse I method now allows three-color staining (two surface markers and BrdUrd), analyzed in a one laser-cytometer for the study of the phenotype of cycling cells, and of their progeny, in vivo and in cell cultures. It also allows the quantitative analysis of cell surface receptor densities in conditions similar to fresh cells. © 1993 Wiley-Liss, Inc.