High throughput image cytometry micronucleus assay to investigate the presence or absence of mutagenic effects of cold physical plasma

Abstract

Promising cold physical plasma sources have been developed in the field of plasma medicine. An important prerequisite to their clinical use is lack of genotoxic effects in cells. During optimization of one or even different plasma sources for a specific application, large numbers of samples need to be analyzed. There are soft and easy-to-assess markers for genotoxic stress such as phosphorylation of histone H2AX (γH2AX) but only few tests are accredited by the OECD with regard to mutagenicity detection. The micronucleus (MN) assay is among them but often requires manual counting of many thousands of cells per sample under the microscope. A high-throughput MN assay is presented using image flow cytometry and image analysis software. A human lymphocyte cell line was treated with plasma generated with ten different feed gas conditions corresponding to distinct reactive species patterns that were investigated for their genotoxic potential. Several millions of cells were automatically analyzed by a MN quantification strategy outlined in detail in this work. Our data demonstrates the absence of newly formed MN in any feed gas condition using the atmospheric pressure plasma jet kINPen. As positive control, ionizing radiation gave a significant 5-fold increase in micronucleus frequency. Thus, this assay is suitable to assess the genotoxic potential in large sample sets of cells exposed chemical or physical agents including plasmas in an efficient, reliable, and semiautomated manner. Environ. Mol. Mutagen. 59:268-277, 2018. © 2018 Wiley Periodicals, Inc.

Combination of Gas Plasma and Radiotherapy Has Immunostimulatory Potential and Additive Toxicity in Murine Melanoma Cells in Vitro

“…strand breaks (DSBs) as we and others have shown before [17,18]. To this end, we analyzed the number of micronuclei in the mono and combination treatments microscopically ( Figure 3I).…”

“…Yet, the images were void of signal saturation, giving accurate results at least in technical aspects. Ionizing radiation is known to induce DNA double-strand breaks (DSBs) as we and others have shown before [17,18]. To this end, we analyzed the number of micronuclei in the mono and combination treatments microscopically ( Figure 3I).…”

Plasma Medicine: A Field of Applied Redox Biology

“…Indeed, a huge number of in vitro studies report on potential genotoxic CAP effects on isolated, naked, or cellular DNA (118). However, several investigations of potential genotoxic effects of CAP by in vitro standard procedures for mutagenicity testing of chemical substances has demonstrated no extended mutation rate of plasma treated cells (119)(120)(121)(122)(123). The main conclusion is that CAP treatment causes no enhanced genotoxic risk.…”

Activation of Murine Immune Cells upon Co-culture with Plasma-treated B16F10 Melanoma Cells

“…Plasma treatment was performed using an atmospheric pressure non-thermal plasma released by the plasma jet kINPen (neoplas, Germany), which was shown to exert no mutagenic effects [37]. As feed gas, argon (99.9999% pure; Air Liquide, France) was used with a gas flow rate of 3 standard liters per minute (slm).…”