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">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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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22.2K Views.  Luminex Corporation. 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.

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

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 ...

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic macrocycles that exhibit differential cytostatic and cytotoxic properties in specific cell lines, depending on the identities of the chelated metal and functional groups. The unique behavior of functionalized corroles towards specific cell lines introduces the possibility of targeted chemotherapy.
Many anticancer drugs are evaluated by their ability to inhibit RNA transcription. Here we present a step-by-step protocol for RNA transcription in the presence of known and potential inhibitors. The evaluation of the RNA products of the transcription reaction by gel electrophoresis and UV-Vis spectroscopy provides information on inhibitive properties of potential anticancer drug candidates and, with modifications to the assay, more about their mechanism of action.
Little is known about the molecular mechanism of action of corrole cytotoxicity. In this experiment, we consider two corrole compounds: gallium(III) 5,10,15-(tris)pentafluorophenylcorrole (Ga(tpfc)) and freebase analogue 5,10,15-(tris)pentafluorophenylcorrole (tpfc). An RNA transcription assay was used to examine the inhibitive properties of the corroles. Five transcription reactions were prepared: DNA treated with Actinomycin D, triptolide, Ga(tpfc), tpfc at a [complex]:[template DNA base] ratio of 0.01, respectively, and an untreated control.
The transcription reactions were analyzed after 4 hr using agarose gel electrophoresis and UV-Vis spectroscopy. There is clear inhibition by Ga(tpfc), Actinomycin D, and triptolide.
This RNA transcription assay can be modified to provide more mechanistic detail by varying the concentrations of the anticancer complex, DNA, or polymerase enzyme, or by incubating the DNA or polymerase with the complexes prior to RNA transcription; these modifications would differentiate between an inhibition mechanism involving the DNA or the enzyme. Adding the complex after RNA transcription can be used to test whether the complexes degrade or hydrolyze the RNA. This assay can also be used to study additional anticancer candidates.

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles

Gallium(III) 5,10,15-(tris)pentafluorophenylcorrole and its freebase analogue exhibit low micromolar cell cytotoxicity. This manuscript describes an RNA transcription reaction, imaging RNA with an ethidium bromide-stained gel, and quantifying RNA with UV-Vis spectroscopy, in order to assess transcription inhibition by corroles and demonstrates a straightforward method of evaluating anticancer candidate properties.

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Ovarian Cancer Detection Using Photoacoustic Flow Cytometry

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies for the detection of CTCs include flow cytometry, microfluidic devices, and real-time polymerase chain reaction (RT-PCR). Despite recent advances, methods for the detection of early ovarian cancer metastasis still lack the sensitivity and specificity required for clinical translation. Here, a novel method is presented for the detection of ovarian circulating tumor cells by photoacoustic flow cytometry (PAFC) utilizing a custom three dimensional (3D) printed system, including a flow chamber and syringe pump. This method utilizes folic acid-capped copper sulfide nanoparticles (FA-CuS NPs) to target SKOV-3 ovarian cancer cells by PAFC. This work demonstrates the affinity of these contrast agents for ovarian cancer cells. The results show NP characterization, PAFC detection, and NP uptake by fluorescence microscopy, thus demonstrating the potential of this novel system to detect ovarian CTCs at physiologically relevant concentrations.

A protocol is presented to detect circulating ovarian tumor cells utilizing a custom-made photoacoustic flow system and targeted folic acid-capped copper sulfide nanoparticles.

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These issues represent drawbacks in workflows that can ultimately result in irreproducibility of research findings. Manual-based workflows are further limiting the advancement and widespread adoption of viscous materials, such as hydrogels used for biomedical applications. These challenges can be overcome by using automated workflows with standardized mixing processes to increase reproducibility. In this study, we present step-by-step instructions to use an open source protocol designer, to operate an open source workstation, and to identify reproducible mixtures. Specifically, the open source protocol designer guides the user through the experimental parameter selection and generates a ready-to-use protocol code to operate the workstation. This workstation is optimized for pipetting of viscous materials to enable automated and highly reliable handling by the integration of temperature docks for thermoresponsive materials, positive displacement pipettes for viscous materials, and an optional tip touch dock to remove excess material from the pipette tip. The validation and verification of mixtures are performed by a fast and inexpensive absorbance measurement of Orange G. This protocol presents results to obtain 80% (v/v) glycerol mixtures, a dilution series for gelatin methacryloyl (GelMA), and double network hydrogels of 5% (w/v) GelMA and 2% (w/v) alginate. A troubleshooting guide is included to support users with protocol adoption. The described workflow can be broadly applied to a number of viscous materials to generate user-defined concentrations in an automated fashion.

This protocol serves as a comprehensive tutorial for standardized and reproducible mixing of viscous materials with a novel open source automation technology. Detailed instructions are provided on the operation of a newly developed open source workstation, the usage of an open source protocol designer, and the validation and verification to identify reproducible mixtures.

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.