Flow cytometric analysis of two incorporated halogenated thymidine analogues and DNA in a mouse mammary tumor grown in vivo.
A technique was developed for the staining of nuclei for DNA using propidium iodide, and incorporated chlorodeoxyuridine (CldUrd) and iododeoxyuridine (IdUrd) using two monoclonal antibodies that showed negligible cross-reactivity. The mouse mammary solid tumor MCaK was labeled in vivo by intraperitoneal injection of the nucleosides. Tumor cell nuclei were stained after isolation from ethanol-fixed solid tumor tissue and acid denaturation. The Br3 antibody, which specifically recognizes CldUrd, was applied first, followed by indirect staining with goat anti-mouse phycoerythrin. The direct fluorescein isothiocyanate conjugate of the B44 antibody, which specifically recognizes IdUrd, was then applied. In the direct conjugate form this antibody reacted only minimally with CldUrd. The nuclei were then stained with propidium iodide. With this dye combination the coefficients of variations of the DNA histograms were consistently in the 2-4% range. Two other dye combinations were compared. The propidium iodide/phycoerythrin/fluorescein isothiocyanate dye combination was the simplest because of the compatibility with single laser flow cytometry.