Flow cytometric analysis of bromodeoxyuridine-induced micronuclei

Abstract

The effects of DNA substitution by the thymidine analogue 5-bromodeoxyuridine (BrdU) on cell cycle progression and micronucleus induction were studied in different mammalian cell cultures. Simultaneous flow cytometric measurements of DNA content and side scatter of nuclei in Chinese hamster embryo (CHE) cells revealed a concentration-dependent temporary block in the G2/M phase of the first cell cycle. NIH 3T3 cells and human amniotic fluid fibroblast-like cells, on the contrary, did not show any cell cycle disturbances in the presence of BrdU. Micronucleus frequency increased as soon as CHE cells started to divide and reached a plateau when all cells have divided. The height of this plateau was almost equal for 60 and 100 microM BrdU. This saturation of micronucleus induction was due to a saturation of BrdU incorporation into DNA already at a doses of 60 microM as shown by the BrdU/Hoechst quenching technique. Indirect immunofluorescent staining of kinetochores with CREST antibodies revealed that nearly all BrdU-induced micronuclei were kinetochore-negative suggesting the presence of acentric chromosome fragments in these micronuclei. DNA distributions of micronuclei measured by flow cytometry showed several peaks representing micronuclei which contain DNA fragments of defined sizes induced by non-random breakage of chromosomes 1 and X as verified by flow karyotyping and C-banding.

Cytotoxicity and genotoxicity of sewage treatment plant effluents in rainbow trout cells (RTG-2)

“…According to Weller et al (1993), micronuclei (MN) and nuclei (N) can be discriminated from unspecific debris using R 1 and R 4 gates. Likewise, N are discriminated using R 3 and R 6 gates and MN using R 2 and R 5 gates on the basis that MN are 1/ 3e1/10 the size of N, and contain 1e10% of the DNA of G1-phase nuclei (Fig.…”

Fluorescence enhancement of DNA-bound TO-PRO-3 by incorporation of bromodeoxyuridine to monitor cell cycle kinetics

“…Simultaneous staining of DNA with a BrdUrd-nonsensitive fluorochrome such as PI or ethidium bromide (EB) allows analysis of cells in G1-, S-, and G2/M-phases of several cell cycles. The continuous BrdUrd labeling technique, apply-ing the BrdUrd/Hoechst quenching effect, has been used for cell kinetic analysis of initially synchronous cell populations (4)(5)(6)(7)(8)(9)(10)(11)(12) and asynchronously dividing cells (13)(14)(15), resolving the complete history of cell proliferation and representing the heterogeneity of the proliferative status of cell populations. The BrdUrd/Hoechst quenching effect has also been applied for detecting DNA-replicating cells after pulse labeling with BrdUrd by staining cells with HO 33342 and mitramycin (MI), a dye whose fluorescence is enhanced by the incorporation of BrdUrd into DNA (16,17).…”

Flow cytometric detection of micronuclei by combined staining of DNA and membranes

“…Gate 2 was defined in the dot plot SSC against DNA fluorescence, as demonstrated in Figure 2b. Particles with higher scatter intensities were found to be mainly nonspecific debris, and particles with low scatter intensities were found to be mainly MN, as verified by sorting of the respective particles (14,18,22). The dot plot of DNA fluorescence vs. membrane fluorescence was used to define gate 3, as demonstrated in Figures 3a and 5a.…”

“…MN contain genetic material that is lost from the genome of cells during mitosis (10). Because microscopic scoring of MN in some hundreds or thousands of cells is a time-consuming procedure, several attempts have been made to automate MN scoring by image analysis (S,l5,20) or by flow cytometry (4,ll- 14,22). Several years ago, we developed a flow cytometric technique using a suspension of nuclei and MN for flow cytometric measurement of DNA content, enabling us to measure frequency and DNA content of MN (12).…”

“…Several years ago, we developed a flow cytometric technique using a suspension of nuclei and MN for flow cytometric measurement of DNA content, enabling us to measure frequency and DNA content of MN (12). Light scatter intensities (forward and side scatter) were also used for a better discrimination between debris and MN, because these signals give additional information on size and structure of these particles (4, 13,14,22). For the analysis of MN in lymphocytes, a combination of two DNA dyes (HO and EB) was used (lS,19).…”