Name: an automated method to perform the in vitro micronucleus assay using multispectral imaging flow cytometry
Uploaded: 2019-05-13T12:30:00
Duration: 12 min 56 s
Description: Watch this Scientific Journal Video about An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry at JoVE.com

The author thanks Christine Probst (Luminex Corporation) for her efforts in developing previous forms of the data analysis template, as well as Dr. Haley Pugsley (Luminex Corporation) and Dr. Phil Morrissey (Luminex Corporation) for reviewing and editing the manuscript.

The author is employed by Luminex Corporation, the maker of the ImageStream multispectral imaging flow cytometer that was used in this work.

In a recent publication Verma et al. underscored the importance of developing a system that combines the high-throughput advantage of flow cytometry with the data and image storage benefits of image analysis35. The MIFC in vitro MN assay described in this paper satisfies this quotation and has the potential to overcome many of the aforementioned challenges in microscopy and flow cytometry methods. The&#160;protocol described here demonstrates that both cytotoxicity and genotoxicity can be evaluated using MIFC. Sample preparation, cellular staining and data collection are straightforward but there are some critical steps in the protocol that should always be implemented. Addition of potassium chloride (KCl) to the cells is critical to swell the cells, generating separation between the main nuclei. This ensures that the masking algorithm can identify all individual nuclei in BNCs and POLY cells (POLY cells) which is necessary for their enumeration. Additionally, KCL provides separation between nuclei and MN, which is essential for accurate MN masking and quantitation. Furthermore, the use of Formalin following the addition of KCl prevents cells from lysing during centrifugation. The addition of Cytochalasin B causes TK6 cells that have undergone more than one nuclear division to be quite large. As a result, the cytoplasm becomes fragile and can lyse if centrifugation is performed immediately after the addition of KCl. Moreover, it is very important to introduce Hoechst to the sample according to number of cells in the sample and not according to a final concentration. For example, a final concentration of 10 &#181;g/mL of Hoechst will uniformly stain a sample of 1 x 106 cells but may not adequately stain a sample containing 5 x 106 cells and can result in many cells with dimly stained nuclei, making analysis difficult. It is also important to note that Hoechst can be replaced with another DNA dye such as DAPI if the MIFC is equipped with the 405 nm excitation laser or DRAQ5 if the MIFC is equipped with the 488 nm and/or 642nm excitation laser(s). If modifying the nuclear stain, it is critical to titrate the stain in order to find the appropriate concentration for the required/desired laser power.
When collecting data on the MIFC it is important to determine the optimal region boundaries for the Gradient RMS features. The boundaries presented in this protocol may require adjustment due to some slight variations between MIFC instruments. The application of this feature during data collection is essential to ensure that highly focused imagery is captured. If data files contain many blurred or unfocused images, it is probable that the masking algorithms in the analysis software will incorrectly highlight staining artifacts in the blurred areas, leading to a high number of false positive artifacts being scored as MN. Although the image processing techniques described here can be difficult, once an analysis template has been developed in the MIFC software, batch processing allows for data files to be automatically analyzed, eliminating user intervention and therefore, scorer bias. Also, if a cell line other than TK6 cells are used to perform the assay, it will be necessary to modify the masks and region boundaries as the morphological properties (e.g., size) of cells will differ from those of TK6 cells.
The results presented here (Figure 5) show statistically significant increases in MN induction when exposing TK6 cells to various doses of Mitomycin C and Colchicine. Statistically significant increases in the frequency of MN when compared to solvent controls were observed for several doses in both chemicals. In addition, no dose of Mannitol induced a cytotoxicity over 30%, nor a statistically significant increase in the frequency of MN when compared to solvent controls, as expected. The protocol described in this paper using MIFC to perform the in vitro MN assay gives expected results from both positive and negative control chemicals. It is very important to perform a number of experiments using both solvent controls and negative control chemicals to develop baseline values of both the frequency of MN as well as the Cytokinesis Block Proliferation Index (CBPI). For genotoxicity, statistically significant increases in MN frequency are determined through comparison to baseline MN frequencies which must be well-known for the cell type being used. In addition all cytotoxicity calculations are based on the CBPI of the control samples and therefore, baseline rates of MONO, BNCs and POLY cells must be well quantified in controls.
Several limitations and advantages of using MIFC in the context of the MN assay have been described in previous work29,32. The main limitations concern lower MN frequencies when compared to microscopy, which probably results from both the lack of flexibility when implementing the scoring criteria in the analysis software as well as the limited depth of field of the MIFC. Well-contoured masks can be created to accurately identify the main nuclei but MN that are touching (or very close to) the main nuclei might be captured within the BNC mask. Additionally, very small MN that can be rather easily scored using microscopy are probably incorrectly missed when using MIFC due the lower limit on the area parameter of the MN mask to avoid scoring small artifacts. In addition to the difficulties present in image-based data analysis, due to its design, MIFC obtains two dimensional projection images of three dimensional cellular objects. This likely causes some MN to be captured at a different depth of focus that the two main MN, making them appear very dim and un-scorable using masking. Moreover, a small fraction of MN could reside behind one of the two main nuclei, making them impossible to visualize and score. Therefore, considering these difficulties, caution should be used when interpreting significant increases in MN frequency at low doses.
Despite these shortcomings, the MIFC method described here offers several advantages over other techniques. Fenech et al. proposed criteria and guidelines that should be considered when developing automated systems and methodologies for MN assays36. These include, but are not limited to, direct visualization of the main nuclei and cytoplasm, determination of the frequency of MN from various doses of the chemical or agent being tested and the ability to quantitate morphology and determine the position of all nuclei and MN to ensure they are within the cytoplasm. This paper shows that the MIFC method developed to perform the in vitro MN assay satisfies (or possesses the potential to satisfy) these criteria. Specifically, images of the nuclei and MN can be captured by the fluorescent lasers while cytoplasmic imagery can be obtained by using the BF LED. Imagery of cells with normal nuclear morphology can be automatically differentiated from those cells with irregular morphology using a combination of advanced masks and features. The results presented for Colchicine and Mitomycin C (Figure 5) show that both genotoxicity and cytotoxicity can be assessed at various doses when compared to solvent controls and that statistically significant MN frequencies are observed where expected. Furthermore, the OECD Test Guideline 487 recommends scoring 2,000 BNCs per test concentration to assess the presence of MN to determine genotoxicity along with at least 500 cells per test concentration to determine cytotoxicity9; this can take over 1 h using manual microscopy. The protocol and results in this paper show that an average of about 6,000 BNCs, 16,000 MONO cells, and 800 POLY cells were captured and scored per test concentration in about 20 min. The rapid rate of data acquisition and the high numbers of candidate cells scored in such a short time highlight another important advantage of employing MIFC to perform the in vitro MN assay.
While the results presented in this paper are encouraging, they are representative of an early proof-of-concept method. This work should be followed up by more thorough investigation of a larger, more diverse chemical set that covers multiple classes and mechanisms of genotoxicity and cytotoxicity such as those suggested by Kirkland et al.37&#160;Conducting such studies are time consuming and labor intensive, and fall outside of the scope of this paper however, these larger scale studies will provide valuable insight into the ability of the method to reliably identify weakly genotoxic agents. The methodology presented here has not yet been miniaturized to a microwell format, which would allow more rapid and efficient screening across a larger dose range. As such, in its current form, the MIFC-based in vitro MN assay presented here may be best suited for labor-intensive follow-up studies or research into good laboratory practices. However, the method will continue to be optimized and validated, and possesses the potential to allow for increased flexibility in detecting chemical specific events related to morphology, such as aneugen exposure that increases the proportion of cells with non-circular nuclei that are still scorable38. Finally, the MIFC method presents an opportunity to introduce additional biomarkers into the MN assay (e.g. kinetochore staining) to provide a more comprehensive view of the mechanism of MN induction.

The in vitro micronucleus (MN) assay is a commonly used test to assess cytotoxicity and genotoxicity as a screening tool in several fields of study such as chemical and pharmaceutical development as well as human biomonitoring among individuals exposed to various environmental, occupational or lifestyle factors1,2,3. MN&#160;consist of chromosome fragments or whole chromosomes generated during cell division that are not incorporated into one of the two main daughter nuclei. Following telophase, this chromosomal material forms into an individual, rounded body inside the cytoplasm that is separate from either of the main nuclei2. Therefore, MN&#160;are representative of DNA damage and have been used for many years as an endpoint in genotoxicity testing4. The most appropriate method to measure MN is the cytokinesis-block micronucleus (CBMN) assay. Using the CBMN assay, the frequency of MN in binucleated cells (BNCs) can be scored by incorporating Cytochalasin B (Cyt-B) into the sample. Cyt-B allows nuclear division but prevents cellular division and thus, restricts scoring of MN to BNCs that have divided only once5.
Protocols using both microscopy and flow cytometry have been developed and validated and are routinely used to perform the in vitro MN assay6,7,8,9,10,11,12,13,14. Microscopy benefits from being able to visually confirm that MN&#160;are legitimate but is time consuming and prone to variability between scorers15. To address this, automated microscopy methods were developed to scan slides and capture images of nuclei and MN16,17,18,19, but the cytoplasm cannot be visualized, making it difficult to determine if an MN is actually associated with a specific cell. Furthermore, these methods have difficulties identifying polynucleated (POLY) cells (including tri- and quadranucleated cells) which are required for the calculation of cytotoxicity when using Cyt-B9. Flow cytometry methods developed to perform the MN assay employ fluorescence as well as forward and side scatter intensities to identify populations of both the nuclei and MN that have been liberated from the cell following lysis20,21,22. This allows data to be acquired from several thousand cells in a few minutes and permits automated analysis23; however, the inability to visualize the cells makes it impossible to confirm that scored events are genuine. Additionally, lysing the cell membrane inhibits the use of Cyt-B as well as creating a suspension that contains other debris such as chromosome aggregates or apoptotic bodies and there is no way to differentiate these from MN24.
In light of these limitations, Multispectral Imaging Flow Cytometry (MIFC) is an ideal system to perform the MN assay since it combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In MIFC, all cells are introduced into a fluidics system and are then hydrodynamically focused into the center of a flow cell cuvette. Orthogonal illumination of all cells is accomplished through the use of a brightfield (BF) light-emitting diode (LED), a side scatter laser and (at least) one fluorescent laser. Fluorescent photons are captured by one of three (20x, 40x or 60x) high numerical aperture objective lenses and then pass through a spectral decomposition element. Photons are then focused onto a charge-coupled device (CCD) camera to obtain high resolution images of all cells that pass through the flow cell. To avoid blurring or streaking, the CCD operates in time delay integration (TDI) mode which tracks objects by transferring pixel content from row to row down the CCD in synchrony with the velocity of the cell in flow. Pixel information is then collected from the last row of pixels. TDI imaging combined with spectral decomposition allows up to 12 images (2 BF, 10 fluorescent) to be captured simultaneously from all cells passing through the flow cell. All captured imagery is stored in sample-specific data files, permitting analysis to be performed at any time using the MIFC data analysis software. Finally, data files retain the link between cellular images and dots on all bivariate plots. This means that any dot on a traditional bivariate plot can be highlighted and its corresponding BF and fluorescent imagery will be displayed25.
Recently, MIFC-based methods have been developed to perform the MN assay for both triage radiation biodosimetry26,27,28,29,30,31&#160;and genetic toxicology32,33&#160;testing. This work has demonstrated that cellular images of main nuclei, MN and the cytoplasm can be imaged with higher throughput than other methods26. All cell types required for analysis, including MONO cells, BNCs (with and without MN), and POLY cells, can be automatically identified in the MIFC data analysis software, and implementation of the scoring criteria developed by Fenech et al. is accomplished through the use of various mathematical algorithms6,34. Results from biodosimetry showed that dose response calibration curves were similar in magnitude to those obtained from other automated methods in the literature when quantifying the rate of MN per BNC29. Additionally, recent work in toxicology demonstrated that images of MONO cells, BNCs (with and without MN) and POLY cells can be automatically captured, identified, classified and enumerated using MIFC. The protocol and data analysis enabled the calculation of cytotoxicity and genotoxicity after exposing TK6 cells to several clastogens and aneugens32.
The protocol presented in this paper describes a method to perform the in vitro MN assay using MIFC. The sample processing technique used in this work requires less than 2 h to process a single sample and is relatively easy to perform in comparison to other methods. The data analysis in the MIFC analysis software is complicated, but creation of the analysis template can be accomplished in a few hours following the steps outlined in this paper. Moreover, once the template has been created, it can be automatically applied to all collected data without any further work. The protocol outlines all steps required to expose TK6 cells to clastogens and aneugens, describes how to culture, process and stain the cells, and demonstrates how to acquire high resolution imagery using MIFC. Furthermore, this paper illustrates the current best practices for analyzing data in MIFC software to automatically identify and score MONO cells, BNCs, and POLY cells for the purposes of calculating both cytotoxicity and genotoxicity.

<table border="0" cellpadding="0" cellspacing="0" style="width:1012px;" width="1012">
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">15 mL centrifuge tube</td>
			<td style="width:244px;">Falcon</td>
			<td style="width:161px;">352096</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:308px;">Cleanser - Coulter Clenz&#160;</td>
			<td>Beckman Coulter</td>
			<td>8546931</td>
			<td style="width:299px;">Fill container with 200 mL of Cleanser.&#160; https://www.beckmancoulter.com/wsrportal/page/itemDetails?itemNumber=8546931#2/10//0/25/1/0/asc/2/8546931///0/1//0/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Colchicine</td>
			<td>MilliporeSigma</td>
			<td>64-86-8</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Corning bottle-top vacuum filter&#160;</td>
			<td>MilliporeSigma</td>
			<td>CLS430769</td>
			<td style="width:299px;">0.22 um filter, 500 mL bottle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Cytochalasin B</td>
			<td>MilliporeSigma</td>
			<td>14930-96-2</td>
			<td style="width:299px;">5 mg bottle</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:308px;">Debubbler - 70% Isopropanol</td>
			<td>EMD Millipore</td>
			<td>1.3704</td>
			<td style="width:299px;">Fill container with 200 mL of Debubbler.&#160; http://www.emdmillipore.com/US/en/product/2-Propanol-70%25-%28V%2FV%29-0.1-%C2%B5m-filtred,MDA_CHEM-137040?ReferrerURL=https%3A%2F%2Fwww.google.com%2F</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Dimethyl Sulfoxide (DMSO)</td>
			<td>MilliporeSigma</td>
			<td>67-68-5</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Dulbecco&#39;s Phosphate Buffered Saline 1X</td>
			<td>EMD Millipore</td>
			<td>BSS-1006-B</td>
			<td style="width:299px;">PBS Ca++MG++ Free&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Fetal Bovine Serum</td>
			<td>HyClone</td>
			<td>SH30071.03</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="240">
			<td height="240" style="height:240px;width:308px;">Formaldehyde, 10%, methanol free, Ultra Pure</td>
			<td>Polysciences, Inc.</td>
			<td>04018</td>
			<td style="width:299px;">This is what is used for the 4% and 1% Formalin. CAUTION: Formalin/Formaldehyde toxic by inhalation and if swallowed.&#160; Irritating to the eyes, respiratory systems and skin.&#160; May cause sensitization by inhalation or skin contact. Risk of serious damage to eyes.&#160; Potential cancer hazard.&#160; http://www.polysciences.com/default/catalog-products/life-sciences/histology-microscopy/fixatives/formaldehydes/formaldehyde-10-methanol-free-pure/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Hoechst 33342</td>
			<td>Thermo Fisher</td>
			<td>H3570</td>
			<td style="width:299px;">10 mg/mL solution</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Mannitol</td>
			<td>MilliporeSigma</td>
			<td>69-65-8</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">MEM Non-Essential Amino Acids 100X</td>
			<td>HyClone</td>
			<td>SH30238.01</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="200">
			<td height="200" style="height:200px;width:308px;">MIFC - ImageStreamX Mark II</td>
			<td>EMD Millipore</td>
			<td>100220</td>
			<td style="width:299px;">A 2 camera ImageStreamX Mark II eqiped with the 405nm, 488nm, and 642nm lasers was used. http://www.emdmillipore.com/US/en/life-science-research/cell-analysis/amnis-imaging-flow-cytometers/imagestreamx-Mark-ii-imaging-flow-cytometer/VaSb.qB.QokAAAFLzRop.zHe,nav?cid=BI-XX-BDS-P-GOOG-FLOW-B325-0006</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:308px;">MIFC analysis software - IDEAS</td>
			<td>EMD Millipore</td>
			<td>100220</td>
			<td style="width:299px;">The companion software to the MIFC (ImageStreamX MKII)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:308px;">MIFC software - INSPIRE</td>
			<td>EMD Millipore</td>
			<td>100220</td>
			<td style="width:299px;">This is the software that runs the MIFC (ImageStreamX MKII)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Mitomycin C</td>
			<td>MilliporeSigma</td>
			<td>50-07-7</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">NEAA Mixture 100X</td>
			<td>Lonza BioWhittaker</td>
			<td>13-114E</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:308px;">Penicllin/Streptomycin/Glutamine solution 100X</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Potassium Chloride (KCl)</td>
			<td>MilliporeSigma</td>
			<td>P9541</td>
			<td style="width:299px;"></td>
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		<tr height="60">
			<td height="60" style="height:60px;width:308px;">Rinse - Ultrapure water or deionized water</td>
			<td>NA</td>
			<td>NA</td>
			<td style="width:299px;">You can use any ultrapure water or deionized water.&#160; Fill container with 900 mL of Rinse.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">RNase</td>
			<td>MilliporeSigma</td>
			<td>9001-99-4</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">RPMI-1640 Medium 1X</td>
			<td>HyClone</td>
			<td>SH30027.01</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:308px;">Sheath - PBS</td>
			<td>EMD Millipore</td>
			<td>BSS-1006-B</td>
			<td style="width:299px;">This is the same as Dulbecco&#39;s Phosphate Buffered Saline 1X&#160; Ca++MG++ free.&#160; Fill container with 900mL of Sheath.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Sterile water</td>
			<td>HyClone</td>
			<td>SH30529.01</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:308px;">Sterilizer - 0.4-0.7% Hypochlorite</td>
			<td>VWR</td>
			<td>JT9416-1</td>
			<td style="width:299px;">This is assentually 10% Clorox bleach that can be made by deluting Clorox bleach with water.&#160; Fill container with 200 mL of Sterilzer.</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:308px;">System Calibration Reagent - SpeedBead</td>
			<td>EMD Millipore</td>
			<td>400041</td>
			<td style="width:299px;">Each tube holds ~10 mL.&#160; https://www.emdmillipore.com/US/en/life-science-research/cell-analysis/amnis-imaging-flow-cytometers/support-training/XDqb.qB.wQMAAAFLBDUp.zHu,nav&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">T25 flask</td>
			<td>Falcon</td>
			<td>353109</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">T75 flask</td>
			<td>Falcon</td>
			<td>353136</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">TK6 cells</td>
			<td>MilliporeSigma</td>
			<td>95111735</td>
			<td style="width:299px;"></td>
		</tr>
	</tbody>
</table>

an automated method to perform the in vitro micronucleus assay using multispectral imaging flow cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The analysis method outlined in this paper allows for the automatic identification and scoring of BNCs, with and without MN, to calculate genotoxicity. In addition, MONO and POLY cells are also automatically identified and scored to calculate cytotoxicity. Published scoring criteria6,34&#160;that must be adhered to when scoring these events are implemented in the MIFC data analysis software. The results presented here indicate that statistically significant increases in MN frequency with increasing cytotoxicity can be detected following exposure of human lymphoblastoid TK6 cells to well-known MN inducing chemicals (Mitomycin C and Colchicine). Similar results for additional chemicals tested have been demonstrated in a separate publication32. In addition, results from the use of Mannitol show that non-MN inducing chemicals can also be correctly identified using the MIFC method outlined here. The parameters described in the protocol to create all masks, features and region boundaries will likely have to be adjusted if different cell types (e.g. Chinese Hamster cells) are used to perform the assay.
Figure 3 shows four selected panels to identify BNCs (Figure 3A-3D). Shown here is a histogram that enables selection of cells with two nuclei (Figure 3A) and&#160;bivariate plots that enable the selection of BNCs with similar circularity (Figure 3B), similar areas and intensities (Figure 3C) and BNCs that have well-separated, non-overlapping nuclei (Figure 3D) as per the scoring critieria6,34. Figure 3E shows the BF and Hoechst images as well as the BNC and MN masks indicating that BNCs with single or multiple MN can be identified and enumerated. This allows genotoxicity to be calculated by determining the rate of micronucleated BNCs in the final BNC population. Figure 4 shows the application of the Spot Count feature using the POLY mask to identify MONO, TRI and QUAD cells. The number of TRI and QUAD cells can then be summed to obtain the final number of POLY cells (Table 1). This enables cytotoxicity to be calculated by using the formula shown in the protocol. Therefore, each dose point in the experiment can be evaluated by both genotoxicity and cytotoxicity parameters.
Figure 5 shows genotoxicity and cytotoxicity values for the aneugen Colchicine, the clastogen Mitomycin C and for a negative control, Mannitol. For Colchicine (Figure 5A) the 0.02 through 0.05 &#956;g/mL doses produced statistically significant increases in MN frequency, ranging from 1.28% to 2.44% respectively over the solvent control (Table 1). In the case of Mitomycin C&#160;(Figure 5B) the two top doses of 0.4 and 0.5 &#956;g/mL produced statistically significant MN frequencies when compared to solvent controls. These MN frequencies were 0.93% at 0.4 &#956;g/mL and 1.02% at 0.5 &#956;g/mL (Table 2). Finally, for Mannitol (Figure 5C), no doses tested induce a cytotoxicity over 30%, nor did they produce significant increases in MN frequency when compared to solvent controls, as expected (Table 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59324/59324fig1.jpg" /> 
Figure 1: MIFC instrument settings. A screenshot of the MIFC settings as described in section step 7 of the protocol. (A) Setting the 405 nm laser power to 10 mW. (B) Setting the BF channels 1 and 9. (C) Selecting the 60x magnification objective lens. (D) Selecting the slowest flow speed which generates imagery with the highest resolution. (E) Specifying the number of events to be collected to 20,000. (F) Clicking the Load button to begin the sample load process. (G) Clicking the Acquire button to begin acquiring imagery. (H) Clicking the Return button to return any unused sample. (I) Scatterplot of BF Aspect Ratio versus BF Area for the selection of single cells. (J) Scatterplot of Hoechst Gradient RMS versus BF Gradient RMS for the selection of focused cells. (K) Histogram of Hoechst intensity for the selection of DNA positive cells. <a href="https://www.jove.com/files/ftp_upload/59324/59324fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59324/59324fig2.jpg" /> 
Figure 2: Analysis software gating strategy. A screenshot of the gating strategy described in section 9 of the protocol. Regions are shown in sequential order for the identification of binucleated cells (red box), micronuclei (yellow box), and mono- and polynucleated cells (blue box). <a href="https://www.jove.com/files/ftp_upload/59324/59324fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59324/59324fig3.jpg" /> 
Figure 3: Identification and scoring of BNCs with and without MN. (A) Selection of cells that have two distinct nuclei. (B) Identification of binucleated cells (BNCs) that have two highly circular nuclei through the use of the Aspect Ratio Intensity feature. (C) Selection of BNCs that have nuclei with similar areas and intensities. This is accomplished by calculating the ratio of the area of both nuclei and the ratio of the aspect ratio of both nuclei. (D) Use of the Shape Ratio and Aspect Ratio features to identify BNCs that have two well-separated nuclei. (E) The Spot Count feature using the micronucleus (MN) mask demonstrating that BNCs with single or multiple MN can be identified and enumerated. <a href="https://www.jove.com/files/ftp_upload/59324/59324fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59324/59324fig4.jpg" /> 
Figure 4: Identification and scoring of MONO and POLY cells. Use of the spot count feature to identify and enumerate mono-, tri- and quadranucleated cells. Component mask 1 allows the identification of mononucleated cells (top image). Component masks 1 through 3 allows the identification of trinucleated cells (middle image). Component masks 1 through 4 allows the identification of quadranucleated cells (bottom image). This figure has been modified from Rodrigues 201832. <a href="https://www.jove.com/files/ftp_upload/59324/59324fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59324/59324fig5.jpg" /> 
Figure 5: Quantification of cytotoxicity. Cytotoxicity quantified using the cytokinesis block proliferation index (black circles) and genotoxicity quantified using the percentage of MN (clear bars) following a 3 h exposure and 24 h recovery for (A) Colchicine, (B) Mitomycin C and (C) Mannitol. Statistically significant increases in MN frequency compared to controls are indicated by stars (chi-squared test; *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001). All quantities are the average of two replicates at each dose point. This figure has been modified from Rodrigues 201832. <a href="https://www.jove.com/files/ftp_upload/59324/59324fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/59324/59324table1.jpg" /> 
Table 1: The parameters required to calculate cytotoxicity (the number of mono-, bi- and polynucleated cells) and genotoxicity (the number and percentage of micronucleated binucleated cells) for Colchicine. All calculated quantities are the average of two replicates at each dose point.
<img alt="Table 2" src="/files/ftp_upload/59324/59324table2.jpg" /> 
Table 2: The parameters required to calculate cytotoxicity (the number of mono-, bi- and polynucleated cells) and genotoxicity (the number and percentage of micronucleated binucleated cells) for Mitomycin C. All calculated quantities are the average of two replicates at each dose point.
<img alt="Table 3" src="/files/ftp_upload/59324/59324table3.jpg" /> 
Table 3: The parameters required to calculate cytotoxicity (the number of mono-, bi- and polynucleated cells) and genotoxicity (the number and percentage of micronucleated binucleated cells) for Mannitol. All calculated quantities are the average of two replicates at each dose point.
Supplement 1: Full Protocol. <a href="https://www.jove.com/files/ftp_upload/59324/Supplement_1-Full_Protocol.docx" target="_blank">Please click here to download this file.</a>
Supplement 2: Mask List. <a href="https://www.jove.com/files/ftp_upload/59324/Supplement_2-Mask_List.docx" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry at JoVE.com

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

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