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

Abstract

This laboratory has previously reported on the development of a flow cytometry-based method for scoring in vitro micronuclei in mouse lymphoma (L5178Y) cells [Avlasevich et al., Environ. Molec. Mutagen. 47 (2006) 56-66]. With this method, necrotic and mid/late stage apoptotic cells are labeled with the fluorescent dye ethidium monoazide. Cells are then washed, stripped of their cytoplasmic membranes, and incubated with RNase plus a pan-nucleic acid dye (SYTOX Green). This process provides a suspension of free nuclei and micronuclei that are differentially stained relative to chromatin associated with dead/dying cells. The current report extends this line of investigation to include the human cell line TK6. Additionally, methods are described that facilitate simultaneous quantitative analysis of cytotoxicity, perturbations to the cell cycle, and what we hypothesize is aneuploidization. This comprehensive cytogenetic damage assay was evaluated with the following diverse agents: etoposide, ionizing radiation, methyl methanesulfonate, vinblastine, ethanol, and staurosporine. Cells were harvested after 30 hrs of continuous treatment (in the case of chemicals), or following graded doses of radiation up to 1 Gy. Key findings include the following: (1) Significant discrepancies in top dose selection were found for five of the six agents studied when relative survival measurements were based on Coulter counting versus flow cytometry. (2) Both microscopy-and flow cytometry-based scoring methods detected dose-dependent micronucleus formation for the four genotoxic agents studied, whereas no significant increases were observed for the presumed nongenotoxicants ethanol and staurosporine when top dose selection was based on flow cytometric indices of cytotoxicity. (3) SYTOX and ethidium monoazide fluorescence signals conveyed cell cycle and cell death information, respectively, and appear to represent valuable aids for interpreting micronucleus data. (4) The frequency of hypodiploid nuclei increased in response to each of the genotoxic agents studied, but not following exposure to ethanol or staurosporine. Collectively, these results indicate that a comprehensive assessment of genotoxicity and other test article-induced toxicities can be acquired simultaneously using a simple two-color flow cytometry-based technique.

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

“…The high numbers of cells captured by the ISX illustrate the improvement in statistical robustness that can be achieved in comparison to microscope‐based versions of the assay. In addition, conventional flow cytometry methods that do not use Cyt‐B typically analyze between 20,000 and 40,000 lysed nuclei 30, 31, 61. The number of MONO cells scored from samples treated without Cyt‐B using the ISX method fall roughly in the middle of this range and have the added benefit of associated BF and fluorescent imagery for every cell collected.…”

“…The speed of this method and the need for minimal user intervention are attractive features for toxicology testing. Over the last decade, significant improvements were made to the methodology by Avlasevich et al 30 and Bryce et al 31 to incorporate additional fluorescent dyes in order to remedy the difficulties encountered when differentiating MN, cellular debris, and free chromosomes from mitotic cells. This method was successful and configured into a commercially available kit by Litron Laboratories and several publications employing many well‐known clastogens and aneugens have demonstrated that statistically significant increases in MN frequency can be detected when compared with controls 32, 33.…”

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

“…Subsequent stripping of cytoplasmic membranes and incubation with the pan-nucleic acid dye SYTOX Green (plus RNase) provides a suspension of free nuclei and micronuclei (MN) that are differentially stained relative to chromatin associated with dead/dying cells. Using this method, good agreement between microscopy-and flow cytometry-based scoring was reported for mouse L5178Y and human TK6 cells [12][13].…”

“…Importantly, the measurement is a multi-parametric assessment of cell health, as it requires nuclei to be EMA-negative, and also to exhibit forward and side scatter characteristics of healthy nuclei. Based on our previous work with this endpoint [13], we hypothesize that the Flow-NBR will tend to be a more sensitive indicator of cytotoxicity than most widely utilized methods. Experiments described herein included several additional cytotoxicity assays that served as a basis for evaluating the reliability and sensitivity of the Flow-NBR endpoint.…”

High throughput toxicity screening and intracellular detection of nanomaterials

“…Further modifications use a 96‐well format in conjunction with a robotic auto‐sampling device. This adaptation requires less test material than conventional test methods, and has a greater compatibility with HTS instrumentation 138, 139, 140, 141…”