Flow cytometric analysis of two incorporated halogenated thymidine analogues and DNA in a mouse mammary tumor grown in vivo

Abstract

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 crossreactivity. 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. Key terms: Propidium iodide, chlorodeoxyuridine, iododeoxyuridine There are several potential applications for the simultaneous and specific quantification of two different halogenated nucleoside analogues in double-labeled DNA. These include direct measurement of recruitment, simultaneous determination of growth fraction and cell kinetics, and more accurate measurement of potential doubling time (T&.A number of bromodeoxyuridine (BrdUrd)-reactive monoclonal antibodies are now commercially available. These antibodies bind differently to the other halogenated thymidine analogues, chlorodeoxyuridine (CldUrd) and iododeoxyuridine (IdUrd). Recently, Bakker et al. (2) described a n indirect method for staining nuclei double-labeled with CldUrd and IdUrd. They found that the monoclonal antibody B44 (5) was minimally reactive to CldUrd after incubation in a highsalt buffer. To stain CldUrd specifically, they used an antibody with minimal reactivity to IdUrd, similar to the Br3 antibody described previously by Vanderlaan et al. (8). Their procedure involved indirect staining of both B44 and the anti-CldUrd-specific antibody. The indirect staining, combined with mandatory incubation and wash in a high-salt buffer, makes the procedure relatively long. Moreover, while Bakker et al. (2) demonstrated simultaneous staining of CldUrd and IdUrd in DNA, they did not show any results of three color flow cytometric analysis.We describe here our investigation of different procedures for three-color analysis of two incorporated halogenated thymidine analogues and DNA. We compared three staining techniques, shortened the staining procedure considerably, and adapted the method for solid tumors labeled in vivo. The simplest method involved the dye combination of phycoerythrin (PE)-tagged second antibody to Br3, fluorescein isothiocyanate (F1TC)-tagged B44, and propidium iodide (PI).

Cytometry of DNA replication and RNA synthesis: Historical perspective and recent advances based on “click chemistry”

“…Time-separated short pulse-exposures of cells to two different halogenated DNA precursors such as IdU and CldU, subsequently detected immunocytochemically using Abs labeled with fluorochromes emitting at different wavelengths, was proposed as a new strategy to significantly expand the analysis of the cell cycle kinetics through consecutive cell divisions in a relatively simple flow cytometric assay (26–28). This elegant double-labeling technique for cell cycle kinetic analysis was reminiscent of the autoradiographic approach utilizing short consecutive pulses of cells exposed to 3 H- and 14 C-labeled thymidine.…”

Localization of plastid DNA replication on a nucleoid structure

“…IdU incorporation were detected using the ability of different commercially available ''anti-BrdU'' antibodies to recognize either CldU or IdU Bakker et al, 1992;Manders et al, 1992;Pollack et al, 1993). Prior to the application of the antibodies, the slides were treated at RT for 5 min with alcoholic 0.2 mol/L NaOH, followed by 5 min each of 70% and 100% ethanol and then air dried.…”

“…The replication sites were identified by a technique that utilizes the ability of two commerically available ''anti-BrdU'' antibodies to detect preferentially either CldU (Caltag Ab) or IdU (Becton-Dickinson Ab) (Manders et al, 1992;Pollack et al, 1993). In the double labelling procedure, cultures were pulsed with CldU for 45 min, washed, and chased in unmodified medium, then exposed to IdU for 1.5 h, conditions chosen to minimize pulse time but allow detectable incorporation.…”

“…The initiation and progression of replication foci were examined by labelling DNA, at separate points in time, with two different analogs, chlorodeoxyuridine (CldU) and iododeoxyuridine (IdU), which are immunologically distinguishable (Manders et al, 1992;Pollack et al, 1993). The spatial distribution of sites of plastid DNA replication on the nucleoids was determined by examining sites of 5-bromodeoxyuridine (BrdU) incorporation.…”

“…The spatial distribution of sites of plastid DNA replication on the nucleoids was determined by examining sites of 5-bromodeoxyuridine (BrdU) incorporation. The initiation and progression of replication foci were examined by labelling DNA, at separate points in time, with two different analogs, chlorodeoxyuridine (CldU) and iododeoxyuridine (IdU), which are immunologically distinguishable (Manders et al, 1992;Pollack et al, 1993).…”