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

Abstract

A new staining method is presented for flow cytometric measurement of micronuclei (MN) in cell cultures and human lymphocytes using membranespecific fluorescent dyes in addition to DNA staining. Several Key terms: UV-B-induced micronuclei, automation of micronucleus scoring, flow cytometry, membrane-specific dyesThe measurement of the frequency of micronuclei (MN) induced in cells by ionizing radiation or by treatment with chemicals is widely used for analysis of cytogenetic damage. 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). 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). A remaining problem is that there are regions of low signal intensities, where discrimination of debris and MN is not obvious. Especially in rat hepatocytes used for the testing of cytogenetic damage of chemicals by flow cytometric analysis of MN (4) and in human lymphocytes ( 2 1 ), the overlay of nonspecific debris and MN can still be a major problem for the analysis of MN.We have, therefore, developed a new staining technique for flow cytometric analysis of MN using membrane-specific dyes in addition to the DNA dyes, especially for those cell types where a large amount of nonspecific debris was found after the preparation of a suspension of nuclei and MN. For staining of membranes of nuclei, MN, as well as membrane particles in the nonspecific debris, the lipophilic dyes 2-hydroxyethyl-7,12,17-tris( methoxyethy1)porphycene (HEPn) and 1,6-diphenyl-l,3,5-hexatriene (DPH) were used. It has been shown that these fluorescent dyes are located within the regions of the hydrocarbon bondings of membranes of cells (1,23). Due to their lipophilic character, they have to be dissolved in organic solvents or in the lipid phase of Address reprint requests to J.M. Wessels, GSF-lnst. f i r Biophysikalische Strahlenforschung, Arbeitsgruppe DurcMulLbytometrie, Neuherberg,

Automation of the in vitro micronucleus assay using the Imagestream® imaging flow cytometer

“…One of the original publications by Nüsse and Marx described a method based on ethidium bromide fluorescence and forward and side scatter intensities to differentiate between debris, nuclei, and MN 27. Although this method agreed well with microscopy, difficulties in distinguishing MN from other DNA positive events persisted 28, 29. Through the addition of ethidium monoazide bromine (EMA) which stains only the chromatin of apoptotic or necrotic bodies, MN can now be more confidently identified and differentiated from the main nuclei and other DNA positive events.…”

In vitro micronucleus assay scored by flow cytometry provides a comprehensive evaluation of cytogenetic damage and cytotoxicity

“…These methods are based on image analysis, laser scanning cytometry, or flow cytometry [10][11][12][13][14][15][16][17][18][19][20].…”

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

“…We have previously reported on the development of a scoring system that may enhance the utility of a flow cytometric procedure originally described by Nüsse and colleagues [7][8][9][10][11]. Most importantly, the new method includes an ethidium monoazide bromide (EMA) staining step in order to label the chromatin of necrotic and mid/late stage apoptotic cells.…”