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><tbody><tr><td>15 mL centrifuge tube</td><td>Falcon</td><td>352096</td><td></td></tr><tr><td>Cleanser - Coulter Clenz&amp;nbsp;</td><td>Beckman Coulter</td><td>8546931</td><td>Fill container with 200 mL of Cleanser.&amp;nbsp; https://www.beckmancoulter.com/wsrportal/page/itemDetails?itemNumber=8546931#2/10//0/25/1/0/asc/2/8546931///0/1//0/</td></tr><tr><td>Colchicine</td><td>MilliporeSigma</td><td>64-86-8</td><td></td></tr><tr><td>Corning bottle-top vacuum filter&amp;nbsp;</td><td>MilliporeSigma</td><td>CLS430769</td><td>0.22 um filter, 500 mL bottle</td></tr><tr><td>Cytochalasin B</td><td>MilliporeSigma</td><td>14930-96-2</td><td>5 mg bottle</td></tr><tr><td>Debubbler - 70% Isopropanol</td><td>EMD Millipore</td><td>1.3704</td><td>Fill container with 200 mL of Debubbler.&amp;nbsp; 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><td>Dimethyl Sulfoxide (DMSO)</td><td>MilliporeSigma</td><td>67-68-5</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline 1X</td><td>EMD Millipore</td><td>BSS-1006-B</td><td>PBS Ca++MG++ Free&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum</td><td>HyClone</td><td>SH30071.03</td><td></td></tr><tr><td>Formaldehyde, 10%, methanol free, Ultra Pure</td><td>Polysciences, Inc.</td><td>04018</td><td>This is what is used for the 4% and 1% Formalin. CAUTION: Formalin/Formaldehyde toxic by inhalation and if swallowed.&amp;nbsp; Irritating to the eyes, respiratory systems and skin.&amp;nbsp; May cause sensitization by inhalation or skin contact. Risk of serious damage to eyes.&amp;nbsp; Potential cancer hazard.&amp;nbsp; http://www.polysciences.com/default/catalog-products/life-sciences/histology-microscopy/fixatives/formaldehydes/formaldehyde-10-methanol-free-pure/</td></tr><tr><td>Hoechst 33342</td><td>Thermo Fisher</td><td>H3570</td><td>10 mg/mL solution</td></tr><tr><td>Mannitol</td><td>MilliporeSigma</td><td>69-65-8</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids 100X</td><td>HyClone</td><td>SH30238.01</td><td></td></tr><tr><td>MIFC - ImageStreamX Mark II</td><td>EMD Millipore</td><td>100220</td><td>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><td>MIFC analysis software - IDEAS</td><td>EMD Millipore</td><td>100220</td><td>The companion software to the MIFC (ImageStreamX MKII)</td></tr><tr><td>MIFC software - INSPIRE</td><td>EMD Millipore</td><td>100220</td><td>This is the software that runs the MIFC (ImageStreamX MKII)</td></tr><tr><td>Mitomycin C</td><td>MilliporeSigma</td><td>50-07-7</td><td></td></tr><tr><td>NEAA Mixture 100X</td><td>Lonza BioWhittaker</td><td>13-114E</td><td></td></tr><tr><td>Penicllin/Streptomycin/Glutamine solution 100X</td><td>Gibco</td><td>15070063</td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>MilliporeSigma</td><td>P9541</td><td></td></tr><tr><td>Rinse - Ultrapure water or deionized water</td><td>NA</td><td>NA</td><td>You can use any ultrapure water or deionized water.&amp;nbsp; Fill container with 900 mL of Rinse.</td></tr><tr><td>RNase</td><td>MilliporeSigma</td><td>9001-99-4</td><td></td></tr><tr><td>RPMI-1640 Medium 1X</td><td>HyClone</td><td>SH30027.01</td><td></td></tr><tr><td>Sheath - PBS</td><td>EMD Millipore</td><td>BSS-1006-B</td><td>This is the same as Dulbecco&#039;s Phosphate Buffered Saline 1X&amp;nbsp; Ca++MG++ free.&amp;nbsp; Fill container with 900mL of Sheath.</td></tr><tr><td>Sterile water</td><td>HyClone</td><td>SH30529.01</td><td></td></tr><tr><td>Sterilizer - 0.4-0.7% Hypochlorite</td><td>VWR</td><td>JT9416-1</td><td>This is assentually 10% Clorox bleach that can be made by deluting Clorox bleach with water.&amp;nbsp; Fill container with 200 mL of Sterilzer.</td></tr><tr><td>System Calibration Reagent - SpeedBead</td><td>EMD Millipore</td><td>400041</td><td>Each tube holds ~10 mL.&amp;nbsp; https://www.emdmillipore.com/US/en/life-science-research/cell-analysis/amnis-imaging-flow-cytometers/support-training/XDqb.qB.wQMAAAFLBDUp.zHu,nav&amp;nbsp;</td></tr><tr><td>T25 flask</td><td>Falcon</td><td>353109</td><td></td></tr><tr><td>T75 flask</td><td>Falcon</td><td>353136</td><td></td></tr><tr><td>TK6 cells</td><td>MilliporeSigma</td><td>95111735</td><td></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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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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Nanyang Technological University

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22.3K Views.  Luminex Corporation. De op beeldstroom gebaseerde micronucleustest kan verschillende beperkingen van methoden overwinnen, zoals geautomatiseerde microscopie en conventionele stroomcytometrie, waaronder lage doorvoer en gebrek aan visuele bevestiging van gebeurtenissen. Het belangrijkste voordeel van deze techniek is dat alle vereiste gebeurtenissen om genotoxiciteit en cytotoxiciteit te bepalen automatisch kunnen worden afgebeeld, geïdentificeerd en gescoord zonder de noodzaak om microscoopdia's te maken. Voor c...

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

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

Cancer Research

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Immunology and Infection

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...

Automation of the Micronucleus Assay Using Imaging Flow Cytometry and Artificial Intelligence

The micronucleus (MN) assay is used worldwide by regulatory bodies to evaluate chemicals for genetic toxicity. The assay can be performed in two ways: by scoring MN in once-divided, cytokinesis-blocked binucleated cells or fully divided mononucleated cells. Historically, light microscopy has been the gold standard method to score the assay, but it is laborious and subjective. Flow cytometry has been used in recent years to score the assay, but is limited by the inability to visually confirm key aspects of cellular imagery. Imaging flow cytometry (IFC) combines high-throughput image capture and automated image analysis, and has been successfully applied to rapidly acquire imagery of and score all key events in the MN assay. Recently, it has been demonstrated that artificial intelligence (AI) methods based on convolutional neural networks can be used to score MN assay data acquired by IFC. This paper describes all steps to use AI software to create a deep learning model to score all key events and to apply this model to automatically score additional data. Results from the AI deep learning model compare well to manual microscopy, therefore enabling fully automated scoring of the MN assay by combining IFC and AI.

The micronucleus (MN) assay is a well-established test for quantifying DNA damage. However, scoring the assay using conventional techniques such as manual microscopy or feature-based image analysis is laborious and challenging. This paper describes the methodology to develop an artificial intelligence model to score the MN assay using imaging flow cytometry data.

The micronucleus (MN) assay is used worldwide by regulatory bodies to evaluate chemicals for genetic toxicity. The assay can be performed in two ways: by ...

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.&#160;We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.
We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation.&#160;Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.&#160;Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters &#8220;aspect ratio&#8221; of a cell in the bright-field channel that aids in the recognition of elongated cells and &#8220;delta centroid XY Ter119/Draq5&#8221; that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate.&#160;The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

Biology

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome and are recycled. During autophagy, cytoplasmic LC3 protein is lipidated and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to form the autolysosome, where the breakdown of the autophagosome vesicle and its contents occurs. The ubiquitin-associated protein p62, which binds to LC3, is also used to monitor autophagic flux. Cells undergoing autophagy should demonstrate the co-localization of p62, LC3, and lysosomal markers. Immunofluorescence microscopy has been used to visually identify LC3 puncta, p62, and/or lysosomes on a per-cell basis. However, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, multispectral imaging flow cytometry was used along with an analytical feature that compares the bright detail images from three autophagy markers (LC3, p62 and lysosomal LAMP1) and quantifies their co-localization, in combination with LC3 spot counting to measure autophagy in an objective, quantitative, and statistically robust manner.

Here, multispectral imaging flow cytometry with an analytical feature that compares bright detail images of 3 autophagy markers and quantifies their co-localization, along with LC3 spot counting, was used to measure autophagy in an objective, quantitative, and statistically robust manner.

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via ...

Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and cell debris, through bulk endocytosis. This pathway plays an essential role in a variety of cellular processes, including the regulation of immune responses and cancer cell metabolism. Given this importance in biological function, examining cell culture conditions can provide valuable information by identifying regulators of this pathway and optimizing conditions to be employed in the discovery of novel therapeutic approaches. The study describes an automated imaging and analysis technique using standard laboratory equipment and a cell imaging multi-mode plate reader for the rapid quantification of the macropinocytic index in adherent cells. The automated method is based on the uptake of high molecular weight fluorescent dextran and can be applied to 96-well microplates to facilitate assessments of multiple conditions in one experiment or fixed samples mounted onto glass coverslips. This approach is aimed at maximizing reproducibility and reducing experimental variation while being both time-saving and cost-effective.

Automated assays using multi-well microplates are advantageous approaches for identifying pathway regulators by allowing the assessment of a multitude of conditions in a single experiment. Here, we have adapted the well-established macropinosome imaging and quantification protocol to a 96-well microplate format and provide a comprehensive outline for automation using a multi-mode plate reader.

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and ...

Extraction and Identification of Micronuclei from Human Peripheral Blood Lymphocytes

A micronucleus (MN) is an abnormal nuclear structure that forms in cells, particularly bone marrow or blood cells, when exposed to external damage, such as radiation, due to unresolved DNA damage or mitotic errors. Once formed, MNs can actively contribute to various carcinogenic processes, including inflammatory signaling and chromosomal genetic rearrangements. MNs contain nuclear DNA, histones, nucleoprotein fragments, and other active proteins, which are closely associated with their functions. Studying the formation and components of MNs is crucial for understanding their role in driving carcinogenic processes. The extraction and purification of MNs are essential to achieving these research objectives. However, the instability of the MN nuclear membrane and its susceptibility to rupture make these processes technically challenging. Currently, only a few studies have reported the use of density gradient centrifugation for MN separation. This study summarizes and simplifies the processes of MN separation and purification. Human peripheral blood lymphocytes exposed to radiation were isolated, and MNs were separated and purified using sucrose buffers of different concentrations in a two-step process. The integrity and purity of the MNs were verified, providing a clear and practical demonstration of the experimental procedure for researchers investigating the causes and functions of MNs.

This protocol outlines a method for extracting and purifying micronuclei from human lymphocytes using sucrose density gradient centrifugation. It provides an experimental basis for investigating the composition and function of micronuclei.

A micronucleus (MN) is an abnormal nuclear structure that forms in cells, particularly bone marrow or blood cells, when exposed to external damage, such ...

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

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