INSTRUCTIONS Thermo Scientific ToxInsight Micronucleus Cartridge High-Content Imaging Reagents Number R043001MN Description Thermo Scientific ToxInsight Micronucleus Cartridge, sufficient materials for 6 96 wells 0.0 Kit Contents: R043001MN Cellular Dye 35 µl Cytokinesis Blocking Agent 6 x 100 µg DAPI Dye 50 µl Wash Buffer (10X) 100 ml Permeabilization Buffer (10X) 100 ml Storage: Upon receipt, store Cellular Dye at -20 C. Store remaining kit components at 4 C. Keep vials containing Cellular and DAPI dyes protected from light. Allow buffers to warm to room temperature before use. See the Solution Preparation section for storage and stability of prepared solutions. Please completely read these instructions and the accompanying material safety data sheets before using this product. The Cytokinesis Blocking Agent (cytochalasin B) is a toxin. The Cellomics Reagents are not for diagnostic use in humans or animals. Introduction The micronucleus assay is an important component of genetic toxicology screening programs (1-3). Micronuclei arise from acentric chromosomes and lagging whole chromosomes (4, 5). Chromosome mutations of both structure and number are implicated in many human diseases. There is substantial evidence that chromosome mutations and related events in oncogenes and tumor suppressor genes of somatic cells are involved in induction and/or progression of some cancers in humans and experimental animals. Thus, the purpose of the in vitro micronucleus assay is to detect those agents that modify chromosome structure and segregation in such a way as to lead to induction of micronuclei in interphase cells. Traditional quantitative assessment of micronucleus induction in 1,000 or more cells per slide is a labor-intensive, manual task. The in vitro micronucleus assay uses cultures of established cell lines, cell strains, or primary cell cultures. The cells used are selected based on their ability to grow in culture and their spontaneous micronuclei frequency. Analysis of micronuclei induction in human lymphocyte cultures indicates that the most convenient stage to score micronuclei is during the binucleate, interphase stage (6, 7). Such cells have completed one cell division after chemical treatment and are therefore capable of expressing micronuclei. Treating cultures with an inhibitor of actin polymerization (cytochalasin B) results in the trapping of cells at the binucleate (or multinucleate) stage where they can be easily identified (Figure 1) (6, 7). Measuring the Cytokinesis-Block Proliferation Index, in addition to %Cytostasis within a culture, provides a simple method of measuring treatment toxicity (8). The Thermo Scientific ToxInsight Micronucleus Cartridge contains optimized reagents for the detection and quantitation of micronuclei in multinucleate cells. The kit provides reagents for fluorescence detection of micronuclei and nuclei and their cellular domains upon treatment of cells grown on collagen-i coated microplates. This assay was optimized and run on the Thermo Scientific ToxInsight Reader using the ToxInsight Micronucleus Cartridge. By incorporating the cartridge with the Thermo Scientific Micronucleus Genotoxicity Panel Template, one can quickly assess the cytotoxicity and genotoxicity of the compounds being studied.
Figure 1. Principle of micronucleus induction using the cytokinesis-block method and the Micronucleus Cartridge. Although rare, spontaneous micronuclei can occur; however by adding cytochalasin B (after adding a genotoxic agent), cells capable of expressing genotoxin-induced MN can be evaluated. Several outcomes are possible: apoptotic cells, arrested cells, cells blocked in cytokinesis (binucleate cells), or cells blocked with the induction of micronuclei (spontaneous or induced). Figure 2. CHO-K1 treated for 3 hours with 500 ng/ml MMC. Left: DAPI stained nuclei/micronuclei. Arrows point to micronuclei in mononucleate, binucleate, and multinucleate cells. Right: Cells stained with the Cellular Dye. 2
Additional Materials Required CHO-K1 cells (ATCC, Product No. CCL-61) Culture Media (F-12K Complete Media) o F-12 Kaighn s Modification (HyClone, cat.# SH30526) o Fetal Bovine Serum (HyClone, cat.# SH30071), final concentration 10% o Penicillin/Streptomycin (HyClone, cat.# SV30010), final concentration 1% Trypsin-EDTA mixture (HyClone, Product No. SH30042) Genotoxic Agents: o Clastogen: mitomycin C (CAS 50-07-7; EMD Biosciences, Product No. 47589) o Aneugen: etoposide (CAS 33419-42-0; Sigma, Product No. E1383) Dimethyl Sulfoxide (DMSO; suggested vendor Sigma #D2438, CAS 76-68-5) Microplates, black, collagen-i coated (Nunc, Product No. 152036) Formaldehyde (37%) (Fisher, Product No. F79-500) 96-well deep-well plate (Nunc, Product No. 278743) Cell Preparation Information Care should be taken to avoid conditions that would lead to positive results that do not reflect intrinsic mutagenicity and may arise from changes in ph, osmolality, or high levels of cytotoxicity. Proper cell culture techniques, including creating and maintaining cryogenic cell stocks and cell passaging, is critical for ensuring the optimal performance of the assay. Thermo Scientific provides guidance on proper cell culture techniques that should be adhered to for the purposes of this assay. Please see Culturing and Use of CHO-K1 Cells in High Content Imaging Assays and Cryopreservation of Mammalian Cell Lines for more information. These and other supplemental information can be found on our website at www.thermoscientific.com/cellomics. We recommend that you review these materials prior to the start of the assay. General instructions are highlighted below. Protocol optimized for CHO-K1 cells. These cells have a doubling time of approximately 14 hours. Culture CHO-K1 cells in F-12K Complete Medium. Split cells when they reach 70-80% confluency (every 2-3 days) at a dilution of approximately 1:10 to 1:40. Do not allow the cells to exceed 80% confluency. Please refer to Culturing and Use of CHO-K1 Cells in High Content Imaging Assays for information on seeding densities. For plating cells, harvest cells with trypsin-edta mixture, dilute into F-12K Complete Medium, and determine cell density. Adjust cell density to approximately 2 10 4 cells/ml in F-12K Complete Medium and add 100 µl of the cell suspension to each well of a 96-well collagen-i coated microplate. Incubate 18-24 hours at 37 C in 5% CO 2. 3
ToxInsight Micronucleus Cartridge Protocol A. Solution Preparation (per 96-well plate). It is highly recommended to run plates in duplicate. Dilution Media/ Vehicle Control Positive Control(s): Clastogen Samples Aneugen Cytokinesis Blocking Agent (cytochalasin B) - Stock Solution Cytokinesis Blocking Agent - Working Solution Cellular Dye Solution 1X Wash Buffer Fix Solution Combine F-12K Complete Medium with the appropriate vehicle for use in making serial dilutions and as a vehicle control for the assay. We recommend that compounds be prepared in DMSO and the maximal concentration of DMSO (not to exceed 1%) be used in all wells. Mitomycin C (MMC): Reconstitute MMC to 500 µg/ml (1.5 mm) in DMSO (Refer to the manufacturers instructions for appropriate storage conditions). For Short Exposure, add 2 µl of stock concentration to 2 ml of F-12K Complete Medium for a final concentration of 500 ng/ml (1.5 µm). For Long Exposure, add 0.4 µl of stock concentration to 2 ml of F-12K Complete Medium for a final concentration of 100 ng/ml (300 nm). Prepare just before each assay. It is suggested that this compound be used as Positive Control 1 and added to column 12 of the assay plate. Etoposide: Reconstitute etoposide to 100 mm in DMSO (Refer to the manufacturers instructions for appropriate storage conditions). For Short Exposure, dilute stock concentration to 1mM and add 3 µl of dilution to 2 ml of Complete Medium for a final concentration of 1.5 µm. For Long Exposure, dilute stock concentration to 1mM and add 2 µl of dilution to 2 ml of Complete Medium for a final concentration of 1 µm. Prepare just before each assay. It is suggested that this compound be used as Positive Control 2 and added to column 11 of the assay plate. We recommend that sample compounds should be prepared in DMSO whenever possible. It is suggested to make a 100 mm stock solution; if found to be insoluble, dilute in half until soluble (i.e., test solubility at 50 mm, then 25 mm, etc.). Add 10 µl ethanol to 1 vial of Cytokinesis Block to create a 10 mg/ml stock concentration. Store stock solution at 4 C for up to 6 months. Add 6.6 µl of Cytokinesis Blocking Agent Stock Solution to 11 ml of warm F-12K medium. Prepare just before each assay. Add 1.1 µl Cellular Dye to 11 ml of warm F-12K Complete Medium. Prepare just before each assay. Store remaining stock solution at -20 C. Add 10 ml 10X Wash Buffer to 90 ml ultrapure water. Store diluted solution at 4 C for up to 7 days. Add 1.2 ml 37% formaldehyde, 240 µl 10X Permeabilization Buffer, and 4.0 µl DAPI to 10.6 ml 1X Wash Buffer. Warm to 37 C. Prepare just before each assay. 4
B. Recommended Plate Layout Figure 3 is a representative plate setup for the Micronucleus Cartridge Assay. We suggest that the assay start with test chemicals at a maximal concentration of 100 µm (column 2) and should be serially diluted at a ratio of 1:2 across the plate. Final DMSO concentrations should remain consistent throughout the plate and should not exceed 1%. Vehicle Control (media + DMSO) will be in column 1, Positive Controls in column 12 (if only using one Positive Control), or columns 11 and 12 (if wanting both aneugen and clastogen Positive Controls). A1 A1 Light Gray: (column 1) = Vehicle Control Medium Gray: (column 11) = Positive Control 2 (optional) Black: (column 12) = Positive Control 1 Other: (wells A2-H10) = Sample Compounds four compounds per plate; 1:2 dilutions in duplicate wells (column 2= highest dose) Note: All wells should contain the same final concentration of the vehicle used. Figure 3. Suggested plate setups. We recommend duplicate plates per four compounds with duplicate wells (four sample test chemicals plus controls per plate). If only one positive control is needed, a 10-point DR curve will be done (left); if two positive controls are desired, a 9-point DR curve will then be done (right). 5
Procedure: It is recommended to first test compounds using the Short Exposure protocol. Long Exposure testing can be used for confirmation or to continue testing if a compound is inconclusive or not found to be genotoxic from Short Exposure. In addition, the concentration range of test substances should produce toxicity ranging from little to no cytotoxicity to 50-60% cytotoxicity. Substances that do not achieve these values should be repeated with an adjusted concentration range and/or repeated as a Long Exposure. OECD test guideline 487 (8) provides additional guidance on the proper conditions, including recommended concentration ranges, adjustments to those ranges, and treatment schedules. Note: Protocol requires approximately 5 hours of hands-on time to perform over a 4 day period (including initial plating). Short Exposure: 1. Thaw and passage CHO-K1 cells as described in the Cell Preparation section and supplemental instructions. Plate cells at 2000 cells/100 µl media /well into each well of a 96-well Nunc Black Collagen I plate (cell culture plate). Incubate at 37 o C in 5% CO 2 for 18-24 hours. 2. Prepare compounds in a 96-well deep-well plate. After incubation, remove media from plates and transfer 100 µl from the compound plate to the cell culture plate and incubate for 3 hours at 37 o C in 5% CO 2. 3. After 3 hours incubation, remove compound media and wash 3x with 100 µl of Complete Media, leaving the final wash in the wells. Return to the incubator for 17 hours. 4. Remove media and add 100 µl of Cytokinesis Block Working Solution to the entire plate. Return to the incubator for 28 hours. 5. After 28 hours, remove Cytochalasin Block Working Solution, add 100 µl of Cellular Dye to entire plate and return to the incubator for 30 minutes. 6. Remove Cellular Dye and wash once with 100 µl of Complete Media. Add 100 µl of Fixation Solution and incubate in fume hood at room temperature for 20 minutes. 7. Aspirate Fixation Solution and wash twice with 100 µl of 1X Wash Buffer, leaving the last wash in the wells. 8. Seal plate and evaluate on the Thermo Scientific ToxInsight IVT Reader with the appropriate Micronucleus protocol, 20x objective, and plate form factor (objective correction collar set to 0.17 for Nunc Black Collagen-1 plates). Long Exposure: 1. Thaw and passage CHO-K1 cells as described in the Cell Preparation section and supplemental instructions. Plate at 2000 cells/100 µl media /well into each well of a 96 well Nunc Black Collagen I plate (cell culture plate). Incubate at 37 o C in 5% CO 2 for 18-24 hours. 2. Prepare compounds in a 96-well deep well plate. After incubation, remove media from plates and transfer 100 µl from the compound plate to the cell culture plate and incubate for 20 hours at 37 o C in 5% CO 2. 3. After incubation, remove compound media, wash 3X with 100 µl of Complete Media, and add 100 µl of Cytokinesis Block Working Solution to entire plate. Return to incubator for 28 hours. 4. After 28 hours, remove Cytochalasin Block Working Solution, add 100 µl of Cellular Dye to entire plate and return to the incubator for 30 minutes. 5. Remove Cellular Dye and wash once with 100 µl of Complete Media. Add 100 µl of Fixation Solution and incubate in fume hood at room temperature for 20 minutes. 6. Aspirate Fixation Solution and wash twice with 100 µl of 1X Wash Buffer, leaving the last wash in the wells. 7. Seal plate and evaluate on the Thermo Scientific ToxInsight IVT Reader with the appropriate Micronucleus protocol, 20x objective, and plate form factor (objective correction collar set to 0.17 for Nunc Black Collagen-1 plates). Note: Store sealed plates in the dark at 4 C. The Cellular Dye should last at least 72 hours post-fixation and ideally should be run within this time period. 6
C. Data Analysis *NOTE: The data acquired using the ToxInsight Micronucleus Cartridge should be analyzed with the Thermo Scientific AddIn for Microsoft Excel and Excel Template for the Micronucleus Assay for accurate prediction. Please see the Thermo Scientific Micronucleus Genotoxicity Panel Assay Guide for more in-depth information on the templates. 1. The Thermo Scientific AddIn for Microsoft Excel and a copy of the Micronucleus Assay Excel template are installed on the computer connected to the ToxInsight instrument. 2. Open the Microsoft Excel 2010 software. On the Excel 2010 Ribbon, click the File tab and then click the New option on the sidebar below the tab. 3. Click on the My Templates option under Available Templates. 4. A window with available templates will open. Click on the Thermo tab. 5. Select the appropriate template. The Excel template file will load. The.xltx extension indicates a template file. a. Select MNGP_CHOK1.xltx. 6. Please follow instructions provided in the Micronucleus Genotoxicity Panel Assay Guide and Excel AddIn User s Guide for additional information on using the Micronucleus Assay template and the Microsoft Excel AddIn tool. 7. Analyze the data according to the instructions. Additional Information A. Dose Response Curves and Performance Data CHO-K1 cells were treated with increasing concentrations of mitomycin C (MMC) for 20 hours. Cells were then washed and treated with Cytokinesis Blocking Agent for 28 hours, stained with the Cellular Dye, and fixed and stained with DAPI Dye. Plates were analyzed using the ToxInsight Reader with the Micronucleus Cartridge. Figure 4. Dose response curve for CHO-K1 cells treated with MMC. The LOED based off of the % MN Frequency and p-value were equivalent for this compound (55.6 ng/ml). The highest concentration of MMC (1500 ng/ml) is invalid because of cellular toxicity. B. Fluorescent Absorption/Emission The approximate absorption/emission maxima of the fluorescent dyes are as follows: Cellular Dye = 540/566 nm DAPI Dye = 345/455 nm 7
C. Recommendations for Automation Plating Cells: To improve the uniformity and throughput of plating cells, use a liquid handling system such as Thermo Scientific Multidrop Combi or WellMate Dispensers. Dead Volumes: Every piece of automation instrumentation has a non-recoverable dead volume associated with it. Be aware of these dead volumes, priming volumes, and rinsing volumes when calculating your reagent requirements. Nonspecific Binding: Because of the potential of reagent interaction with large surface areas inherent to tubing, syringes and peristaltic pumps, pre-priming with reagents or pre-coating with protein blockers may be warranted. Mixing: Gentle mixing may be required when adding a DMSO-based solution to keep overly concentrated solutions from lying on top of the cell layer. Be careful not to dislodge cells or beads during mixing procedures. Cell Washing: Use an automated plate washer designed to gently wash attached cells. Be careful not to dislodge cells or beads during cell washing. Incubation: Minimize the time when plates with live cells are out of a controlled CO 2 environment. For best results, use an automated incubator to deliver plates to a pipetting deck. Exposure: Minimize operator exposure to fixative by some form of containment. Some reagents and compounds are light-sensitive; be aware of these constraints when scaling up for an automated run. Adapting to other plate formats: When using different plate types, adjust reagent volumes as needed. Some suggested starting volumes are listed in Table 1. Table 1. Suggested volumes to use for different cell culture plates. 96-Well Plates 384-Well Plates 24-Well Plates Kit Component (µl/well) (µl/well) (µl/well) Fixation Solution 100 25 400 Wash Buffer 100 25 400 Cellular Dye 100 25 400 Cytokinesis Block 100 25 400 DAPI solution 100 25 400 ToxInsight Protocol for Micronucleus Genotoxicity Panel Template One protocol is provided for the CHO-K1 cell line for use with this Micronucleus Cartridge and the Micronucleus Genotoxicity Panel Template. The protocol is available in the Protocol Manager under the Micronucleus.V4 folder in the ToxInsight IVT Platform Cellomics Scan software. The protocol is: MNGP_CHOK1_2000_3or20hr_Nunc_ColI_2ch References 1. Matter, B., and W. Schmid. 1971. Mutation Res 12:417-25. 2. Heddle, J. 1973. Mutation Res 18:187-90. 3. MacGregor, J., et al. 1983. In Developments in the Science and Practice of Toxicology, eds. A. W. Hayes, R. C. Schnell, and T. S. Miya, 555-58. Amsterdam: Elsevier. 4. Maier, P., and W. Schmid. 1976. Mutation Res 40:325-38. 5. Hayashi, M., et al. 1984. Mutation Res 141:165-69. 6. Fenech, M., and A. Morley. 1985. Cytobios 43:233-46. 7. Fenech, M., and A. Morley. 1985. Mutation Res 147:29-36. 8. OECD. 2010. In vitro Mammalian Cell Micronucleus Test. OECD Guideline for Testing of Chemicals No. 487. Available at: http://iccvam.niehs.nih.gov/suppdocs/feddocs/oecd/oecd-tg487.pdf For Technical Support for the Thermo Scientific Cellomics ToxInsight Cartridges, call 1-800-432-4091 - Ext. -2525 (US Toll Free). For more information, visit www.thermoscientific.com/cellomics or call 800-432-4091 (US Toll Free) or 412-770-2500. 8
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