The study of DNA damage at the chromosomal level is an important part of genotoxicology because chromosomal mutations are an important part of the cancer process. The micronucleus assay is considered one of the best methods for evaluating chromosome damage because of its ability to accurately detect chromosome loss and chromosome breakage. Since micronuclei are only found in cells that have completed nuclear division, such cells can be identified by their binucleation using a method that blocks cytoplasmic cleavage (cytokinesis) with the microfilament polymerization inhibitor cytochalasin B. This inhibitor is used for the detection of cytoplasmic cleavage, but it is also used for the detection of chromosome loss and chromosome breakage. This cytokinesis-blocked micronucleus (C B M N ) assay has good precision because the data obtained are not confounded by altered cell division kinetics caused by cytotoxic agents or by suboptimal cell culture conditions. This approach is now used on a variety of cells to detect genetic damage in populations, to screen for potential genotoxicity of chemicals and for other special purposes such as predicting tumor sensitivity to radiation and interindividual differences in sensitivity to radiation. Applying the current basic research methodology, the CBMN assay can be used for the detection of chromosome breakage, chromosome loss, chromosome reorganization (nucleoplasmic b rid g e , nucleoplasmic bridge), gene amplification (nuclear bud), cytokinesis block, necrosis, and apoptosis by applying simple morphological criteria. The CBMN assay modified with cytarabine was able to detect excision repair damage. The use of molecular probes allows the differentiation of chromosome breaks from chromosome deletions and, more importantly, the detection of nondisjunction chromosomes in non-micronucleated binucleated cells. The CBMN technique thus allows for multiple and complementary genotoxicity and cytotoxicity measurements in a relatively simple system. This chapter describes the basic principles and methods of the CBMN assay (including detailed scoring criteria for all genotoxicity and cytotoxicity indices) and the prospects for its development.
Written by Martin, this experiment is from the "Environmental Genomics Laboratory Guide".
Operation method
Micronucleus assay for the detection of DN A damage at the chromosome level Move I. Materials 1. Cytoplasmic division blocking micronucleus test (1) Serum samples obtained from patients with scleroderma C R E S T subtype. (2) F I T C labeled rabbit anti-human I g G secondary antibody. (3) Peroxidase-labeled rabbit anti-human I g G . (4) Diaminobiphenyl solution (D A B ): I m g /m L dissolved in 0. 5 mol/L Tris alkaline buffer solution, p H 7. 6. (5) NiCl2 solution: 8% solution in 0.5 mol/L Tris alkali buffer, p H 7.6, prepared before use. (6) DAB reaction mixture: I mL DAB solution, 3 mL Tris base buffer solution, p H 7.6, 25NiCl2 solution, 40 uL of 0-Imol/L aqueous imidazole solution and 10 u L of 30% hydrogen peroxide. Prepare before use. (7) Neutral red solution: 0.1 % in double-distilled water. In this assay only M N i appearing in cells that have completed a single nuclear division after stimulation with P H A are used for analysis. These cells can be recognized by the binucleate morphology resulting from the addition of Cyt B prior to the first mitotic division to keep the cells from undergoing subsequent cell divisions. The proportion of binucleated cells in all living cells (all cells except necrotic and apoptotic cells) 72 h after PHA stimulation under optimized conditions can be 3 5 % to 60 % or more. All experimental equipment must be 1.1 Lymphocyte Isolation, Cell Culture, and Cell Collection (1) Collect fresh venous blood in tubes containing the anticoagulant heparin and isolate at 22°C for 4 hours. (2) Dilute the blood with sterilized 0.85 % NaCl at a rate of I : 1 and mix by gentle inversion. (3) Gently cover the diluted blood with a Ficoll Paque (Pharmacia) density gradient at a ratio of approximately 1 : 3 (e.g., 2 mL of Rcoll Paque to 6 mL of diluted blood), taking care not to disturb the interface between the two phases. (4) Centrifuge at 400 g at 22°C for 25~40 min. (5) Lymphocytes are located between Ficoll Paque and diluted plasma. After collection, add 3 to 5 times the volume of HBSS at 22°C. Depending on the volume, centrifuge the resulting cell suspension at 280 to 400 g for 5 to IOmin0 . (6) Discard the supernatant, resuspend the cells with 2 to 5 times the volume of HBSS, and centrifuge at 180 to 400 g for 5 min depending on the volume. (7) Discard the supernatant and resuspend the cells with I mL of RPMI1640 culture medium. (8) Cell concentration was calculated by cell counting, and cell concentration was adjusted according to the percentage of live cells obtained from the Taipan blue assay. (9) Adjust the cell concentration to 0.5 XIO6~I. OXlO6 cells/mL with RPMI1640 culture solution containing 10%~15% inactivated fetal bovine serum, and incubate the cells in a round-bottomed culture tube (10 mM wide) with 0.75~1.0 mL of culture solution. (10) Add 10 ML/mL PHA (Glaxo Wellcome HA1 5 ) to each culture tube to stimulate lymphocyte division. Unscrew the cap and incubate at 37°C, 5 % C0 2 , under humid conditions. The concentration of PHA should be optimized according to the purity and source of the product to ensure that cells produce the maximum number of binucleated cells after blockade with CytB. (11) After 44 h of PHA stimulation, add 4.5 pg of CytB per ml of culture medium (wear gloves and operate in a fume hood): Dissolve IOOmL of CytB reservoir, add 900 of culture medium and mix. Add 75 mL of the mixture to 1 mL of ±priming solution to achieve a final concentration of 4.5 Mg CytB/mL (some laboratories have also successfully used a concentration of 6.0ugCytB/mL). Continue incubation. (12) Twenty-eight hours after the addition of C y t B, collect the cells using a blood cell separator (cytocentrifuge, ShandonElliot). Discard 1,000 uL of culture solution and gently resuspend the cells in a tube. Transfer 100 ~120ul of cell suspension to a hemocyte centrifuge cup (ShandonElliot) and centrifuge until two dots are produced per slide (see Note 1). (Set the cell centrifugation program as follows, Time: 5 min, Rotation speed: I O O fiO Remove the slides from the hemocyte separator, air-dry for 1 0 ~ 2 0 m i n , and fix for l 0m in with 1 0 0 % methanol. (13) Staining with different methods that can distinguish the boundary between nucleus and cytoplasm. Used in our experiments was "DiffQuik", a commercially available finished product that gives rapid, optimized results (see Note 1). (14) After staining the slides are allowed to dry and the coverslips are covered with D P X solution. This step needs to be done in a fume hood and the slides stored in the fume hood until needed for use. Duplicate samples are required for control and genotoxin-treated cell cultures , and slides are prepared for each sample. This is necessary for obtaining standard deviations of experimental data, such as the coefficient of variation (standard deviation), which needs to be labeled for each set of replicates (see Notes 2 and 3). This experimental design is shown in Figure 2. For sudden light microscopy, the recommended staining method is acridine orange (40 ug/mL in Sorensen's Mercy buffer, p H 6 . 9 ) for staining. If a cytocentrifuge is not available, slides can be prepared using the method described below for whole blood cultures 1.2 Examination of Slides and Evaluation of MN Frequency Slides should preferably be viewed with a light microscope or fluorescence microscope at a magnification of 1000 times. Slides should preferably be labeled with the analysis _ code so that the analyst does not know the meaning of the slide (blinding). Analysis of the data requires two different analysts to evaluate each replicate experiment using the same microscope (see Notes 4 and 5). The number of cells to be counted (see Note 6) needs to be determined based on the experiment's expected detection of a change in the MN index and the expected standard deviation. Each slide should be obtained The following information should be obtained for each slide: (1) Count the number of micronuclei (M N i ) within at least 1000 binucleated (B N ) cells and the frequency of M N i within 1000 B N cells. The criteria for counting M N i in B N cells are as follows. (2) Distribution of B N cells containing 0, 1, or more micronuclei (M N i ); the number of M N i in a binucleated cell is approximately 0 to 3 in normal human lymphocytes, but may sometimes be greater than 3 depending on toxicant exposure and age. (3) Frequency of B N cells with micronuclei in at least 1000 B N cells. (4) Count the frequency of nucleoplasmic bridges in 1000 BN cells. The criteria for counting nucleoplasmic bridges in B N cells are as follows. (5) The proportion of mono-, bino-, trinucleated and tetranucleated cells per 500 cells. From this information, the nuclear division index (explained later) can be derived. (6) In addition to the frequency of living mononuclear, binuclear or multinucleated cells, the proportion of dead or dying cells due to apoptosis or necrosis per 500 cells on the same slide is counted (the criteria for this are listed later) (see Note 7). Note that when it is not possible to determine how to categorize a cell, it is best to ignore it. The basic information to be included in a statistical table is shown in Table 1. 1.3 Criteria for Sorting Dinucleated Cells and Counting Micronuclei, Nucleoplasmic Bridges, Nuclear Buds, Apoptotic and Necrotic Cells (1) Sorting criteria for binucleated cells: Cells with blocked cytoplasmic division for MN frequency calculation must meet the following characteristics: a. Cells must be binucleate. b. The two nuclei in a binucleate cell must have intact nuclear membranes and must be in a single cytoplasm. c. The two nuclei in a binucleated cell should be similar in size, coloring shape, and density. d. The two nuclei in a B N cell may be connected by a cytoplasmic bridge not exceeding 1/4 nuclear diameter. e. Two nuclei in a B N cell may be in contact with each other, but should not overlap. Such overlapping binucleated cells should be counted only if the boundary between the two nuclei is well defined. f. The cytoplasmic boundary or cell membrane of a binucleated cell must be intact and clearly distinguishable from nearby cells. The types of binucleated cells that may or may not be used for counting are shown in Figure 3. Types of cells that should not be included in the calculation of micronucleus frequencies include mononuclear, trinuclear, tetranuclear, and multinucleated cells, as well as apoptotic or necrotic cells (Figure 4). Criteria for counting micronuclei: M N i are morphologically similar to nuclei, only smaller. They also have some of the following Characteristics: a. The size of the MNi diameter of human lymphocytes usually accounts for 1/16 to 1/3 of the diameter of the primary nucleus in binucleated cells, and its area is equivalent to 1/256 to 1/9 of the primary nucleus. 1.4 Nuclear Dissociation Index (NDI) and Nuclear Dissociated Cell Toxicity Index (NDCI) (1) Calculate the NDI according to the method of Eastmond and Tucker (30). 500 live cells were counted and the frequency of cells containing one, two, three or four nuclei was calculated using the following formula: NDI = (M 1 + 2 X M 2 + 3 X M 3 + 4 X M 4)/N M l ~ M 4 refers to cells containing 1 to 4 nuclei, and N indicates the total number of viable cells. The N D I index, as well as the proportion of binucleated cells, is a useful parameter for comparing the mitogenic response of lymphocytes and for testing the cytostatic effect of drugs. (2) A more accurate method used to evaluate the state of nuclear division is to include the number of necrotic and apoptotic cells in the total number of cells, since dead cells may account for a large proportion of the total number of cells under the action of high doses of toxic substances. Therefore, it should be noted that if necrotic and apoptotic cells are not included, the proportion of binucleated cells and the N D I index will be high. (3) The following modified equation can be used to more accurately estimate the state of nuclear division and the kinetics of cell division, taking into account both necrotic and apoptotic cells. NDCI= (A P + Nec+ M l+ 2X M2+ 3 X M3+ 4 X M4) /undefined NDCI: nuclear division cytotoxicity index; A p : the number of regulated cells; Nec : the number of necrotic cells; M l ~ M 4 refers to cells with I ~ 4 nuclei; N * indicates the total number of cells (live and dead). In an examination of the M N of human G . phase lymphocytes, it was found that for chemicals and ultraviolet radiation that primarily cause base damage and DNA adducts rather than DNA strand breaks or spindle damage, MN formation was significantly lower compared to cytotoxicity (2 1 ). We speculate that this may be due to efficient repair of the damage or that unrepaired damage sites do not form DNA double-bond breaks after a round of DNA synthesis. Therefore, we hypothesized that inhibition of shear repair with cytarabine (ARA) would cause the base damage to translate into single-strand breaks and then double-bond breaks after DNA synthesis, resulting in the creation of a non-filamentous fragment that would appear as an MN within a single division cycle (21, 31). (1) Based on this concept (Fig. 8), our addition of ARA (1 ug/m L culture medium) during the first 16 h of lymphocyte culture (before DNA synthesis) indeed increased MN formation (10-fold or higher) after UV irradiation or MNU treatment. In contrast, the addition of ARA after X-ray exposure caused only a 1.8-fold change in the amount of MN, because X-ray-induced damage to DNA adducts or bases is less frequent than breakage of DNA chains. This method has been used to identify insecticides that induce shear repair and to distinguish between genetic poisons that do or do not induce shear repair. (2) The A R A assay is an important complement to the C B M N assay, especially when strong cytotoxic effects and weak M N production are observed. (3) D N A shear repair damage can only be accurately detected by the A R A method after the C B M N assay because (a) the transition from D N A shear repair damage to M N occurs only when nuclear division is complete; (b) the addition of A R A may cause a change in the kinetics of cytokinesis, which may interfere with the results of the M N assay in the absence of Cyt B. The A R A assay is an important complement to the C B M N assay, especially when strong cytotoxicity and weak M N production are observed. (4) A R A inhibits D N A polymerase and therefore may cause D N A double-strand breaks in cells undergoing D N A replicative synthesis. Therefore, this method is only useful in P H A-stimulated G . phase lymphocytes, exposure to A R A before G 1 phase, S phase is only possible because shear repair is normally activated in G 1 phase. (5) In practice this means that cells are added to A R A within 16-20 h after P H A stimulation, after which the cells are washed to remove the A R A and cultured in deoxycytidine-containing medium to reverse the inhibition of D N A polymerase by the A R A. (6) The above steps were followed by standard C B M N (as described previously). More detailed procedures and typical results can be found in the articles by Fenech and Nexille ( 2 1 ) and Surrales et al. (3 2 ). 3.1 Whole blood cell culture of lymphocytes (1) Whole blood culture can also be used to perform C B M N experiments on human lymphocytes. (2) - Generally 0-4 to 0-5 m L of whole blood is added to 4.5 m L of culture solution (e.g., RPMI 1640) containing fetal bovine serum, glutamic acid, antibiotics, and PHA. (3) Cyt B is added 44 h after PHA stimulation. The recommended optimal concentration of Cyt B in whole blood cell culture is 6 u g/m L (33). (4) Dinucleated lymphocytes were collected 28 h after addition of Cyt B. The procedure was as follows: a. Collect the cells by centrifugation at 300 g for 5 min and discard the supernatant. b. Hypotonic treatment with 7 ml of 0.075 mol/L KCI pre-cooled at 4°C to lyse erythrocytes, centrifugation at 300 g for 8 m i n Centrifuge at 300 g for 8 m i n . Discard the supernatant and add 5 mL of methanol-acetic acid (3:1) fixative (must be added while shaking to prevent formation of clumps). d. Then centrifuge at 300 g for 8 min and wash twice with fixative. e. Gently resuspend the cells and place a drop of the resuspension on a slide to dry. It is also possible to isolate binucleated lymphocytes directly from whole blood cell cultures using Ficoll gradient and then transfer the cells to slides by centrifugation prior to fixation and staining; this method avoids the need for hypotonic treatment to better preserve the cytoplasm. (5) Staining with 0.1 m ○ l/L potassium phosphate buffer solution (p H I 3 ) containing 1 0 % Jimsa's staining solution for light microscopic use. Alternatively, acridine orange (0.l m o l /L of 0.1 m o l /L potassium phosphate buffer solution containing l O ug/m L, pH 6.9, for use under a fluorescence microscope) was used. 3.2 Mouse lymphocyte culture (1) Lymphocytes are isolated from spleen or peripheral blood and cultured as described by Fenech et al. (34). (2) Because murine lymphocytes have a shorter division cycle than human lymphocytes, it is necessary to add C y t B within i8 h after schizontin stimulation , and to collect the cells 20 h later. Depending on the culture conditions, it is even possible to obtain a better proportion of binucleated cells after 72 h after schizonin stimulation 3.3 Other primary cell cultures including tumor cell cultures CBMN assays can be used for other cell types to detect DNA damage, including in vitro, in vivo, or in vitro assays derived from in vivo. It is most important to remember the following: (1) make sure that the M N i counted is produced in the first nuclear division following the action of the genotoxic agent; and (2) do preexperiments to determine the concentration of Cyt B and the duration of the incubation to obtain the greatest number of dividing cells blocked in the binucleate phase (see Note 10). It is also important to keep in mind that Cyt B may require 6 h to exert its effect of blocking cytoplasmic division (unpublished data). a. When using established cell lines or primary cultured cell lines derived from dividing cell populations, Cyt B is usually added immediately after genotoxicity to obtain all binucleate cells undergoing the first nuclear division - an incubation time of 24-48 h is usually required before collecting the cells, depending on the duration of the cell cycle. b. Adherent cells are first digested and then centrifuged by hematopoietic cell separation as described for human lymphocytes. Specific methods have been described for nucleated bone marrow cells (14), pulmonary fibroblasts (1 5 ), skin keratinocytes (3 6 ), and primary tumor cell cultures (1 3 ). c. To analyze MN induced in vivo, a more operational approach is to block cytoplasmic division of dividing cells by placing the cells in a Cyt B-containing cell culture medium after isolation from the animal. This method has been successfully applied to a variety of cells including fibroblasts, keratinocytes, and nucleated bone marrow cells. (1) There is controversy as to whether Cyt B, which is used to accumulate binucleated cells, affects M N expression (28). Studies in normal cells have not found that CytB induces MNi; nor has CytB shown a dosage effect at concentrations normally used to block cytoplasmic division that causes an increase in the frequency of MN appearance (100, 37-39). Recent studies have suggested that spindle filament toxicant-induced MN expression in cytoplasmic division-blocked BN cells may be less frequent than expected. This is because the shortened distance between the poles increases the chance that lagging chromosome segments or entire chromosomes will return to the nucleus. However, this does not affect the validity of the C B M N experiment (40). (2) Increasingly, vision has been devoted to the development of in vitro M N assays without Cyt B to minimize the effects of Cyt B, but the lack of a cytokinetic control is likely to produce false negatives (e.g., inhibition of nuclear division would prevent M N expression). (3) Although there is a lack of evidence that normal cellular CBMN assays can cause false-positive results, there is much evidence that M N assays that do not take into account nuclear division block cause false-negative results or underestimate MN in human lymphocytes (10, 11, 41), and the shortcomings of M N assays without Cyt B are exemplified in Figure 9. (4) Nevertheless, recent studies comparing MN assays with and without Cyt B have concluded that similar CBMN and MN results can be obtained in the absence of Cyt B in the detection of strong breakers if the cells are in a good state of growth and have appropriate culturing and karyorrhexis (42, 43). (5) Mathematical Modeling of M N Expression Predictions (1 ) M N counting in binucleated cells is the most reliable way to calculate M N frequencies (2 ) M N counting in mononucleated cells without cytokinesis blockage elicits false-negative results when nuclear divisions are significantly blocked by the chemicals tested or when culture conditions do not yield ideally dividing cells (44) (6) Therefore, the M N frequencies obtained with monocytes not treated with Cyt B cannot be used as a conclusive result of the experiment and need to be further confirmed by negative results of the CBMN assay. Taking full advantage of the C B M N assay requires distinguishing between M N i originating from whole chromosomes, and M N i originating from fragments without a mitotic site. This can be accomplished by specific probes against the mitotic site D N A or by specific antibodies against mitotic proteins that accumulate in active chromosomal mitotic regions. For human cells or other cells with heterogeneous chromosome sizes, it is not appropriate to use the size of the M N to make judgments, because a small M N may contain fragments of large chromosomes or an entire small chromosome. The simplest and most economical method is the use of antimitotic antibodies (45), but this method does not discriminate between specific chromosomes and may not detect chromosome loss that occurs by deletion of the mitotic proteins of inactive centromeres (46). Determination of centromeric regions by in situ hybridization (ISH) is costly and laborious but has better specificity; for example, chromosome-specific mitotic probes can be used to detect nondisjunction events (unequal segregation of homologous chromosomes in daughter nuclei) in binucleated cells (17). In this chapter, only the mitogen antibody method is described. For the detection of the centromeric region with 1S H see Farooqi (17), as well as H ando (18), Ehajouji (23, 47), Schuler (1) Collect BN cells by the same method as the standard C B M N experiment, transfer them to slides with a hemocyte centrifuge, air-dry them for 5 m i n , fix them with formaldehyde for IOmin, and air-dry them again. (2) At this step, the slides can be used immediately or stored in a sealed dry box in the vapor phase of liquid nitrogen for a maximum storage period of 3 months. (3) To detect the mitogen on a stored slide remove the slide from liquid nitrogen and equilibrate it in a sealed box at room temperature. 5.1.2 Mitogen Detection (1) Anti-mastocyte serum can be purchased commercially or obtained from immunization clinics that have sera from patients with scleroderma subtype CREST (48). The use of the latter serum requires human ethical approval and the consent of the donor patient. (3) Detection of mitotic proteins with F I T C-labeled secondary antibodies, although more direct, requires a fluorescence microscope and a nonpermanently prepared slide. This synaptic technique has been described in detail elsewhere (45). Another alternative is staining with immunoperoxidase, which results in permanently preserved slides (49), which is more practical as a routine assay and will be described in detail in the next paragraph. (4) In the fluorescence peroxidation method, the fixed slides are placed in a humid chamber containing an anti-matrix protein primary antibody diluted with Tris& buffer (P H 7. 6,6. O g Tris base/L saline solution) at a ratio of 1 : 4 0 , and incubated overnight at 20°C. The slides are then stained with an immunoperoxidase enzyme (49), which is more practical as a routine assay. (5) Prepare a negative control with diluted normal human serum. (6) The next day, the slides were rinsed by submerging them for 30 s in the same Tris saline buffer in which the antibody was diluted. (7) Without drying, the slides were dried and incubated with peroxidase-labeled rabbit anti-human I g G antibody for 3 h . (8) Drain the slides again and prepare for peroxidase histochemistry. (9) The immunohistochemical method that gives the best comparison is the standard D A B reaction improved by imidazolium nickel chloride that produces a black precipitate (50, 51). (10) The D A B reaction is prepared before use and then filtered through 0.22 filter paper to prevent nonspecific precipitates from appearing on the slide. (11) Slides need to be stained in batches, including negative controls. (12) React Imin at 20°C, then stop staining and rinse the slides in water. (13) The slides are then air dried and stained with the nuclear dye neutral red (0.1 % aqueous solution) for about 30s, rinsed with water and air dried to prepare permanent slides. (0.1 % aqueous solution) for about 30s, rinsed in water and air-dried to prepare permanent slides. 5.1.3 Counting procedure (1) Limit the counting of M N i mitotic sites to binucleated cells containing at least 20 mitotic sites per nucleus. (2) Categorize at least 100 M N i by the presence or absence of a mitotic site and indicate the number of mitotic sites each M N contains. (3) Calculate the final ratio of M N i containing mitotic sites using the following formula: (朽一 P c ) / (l-P c ), P c refers to the proportion of M N i that are positive for peroxid Caveat (1) During our CBMN experiments, the most problematic part is in the preparation and staining of slides, because the counting results depend on the quality of the preparation. Major considerations: (a) Cells should be blown gently before placing them on the slide to avoid clumping;(b) Cell density should be moderate so that cytoplasmic boundaries can be easily recognized;(c) Try one slid For more product 
2. Micronucleus mitosis detection
All experimental equipment must meet biosafety requirements to protect the operator and all solutions used in this procedure must be sterilized by filtration.
(25) and others. The various results expected to be detected with different techniques are shown in Fig. 10. 
1 Filling point detection of MNi in CBMN experiments
(2) The serum needs to be detected on a smear of mid-stage dividing cells of cultured cells by observation under a fluorescence microscope with F ITC-labeled rabbit antihuman secondary antibody. Only sera that specifically react with mid-stage chromosome nodes can be used for later experiments.