mouse bone marrow cell micronucleus test
mouse bone marrow cell micronucleus test
The micronucleus test can be used for: chromosome damage and a rapid test method for chemical toxicants that interfere with cell mitosis.
Operation method
mouse bone marrow cell micronucleus test
Principle
The micronucleus test is a rapid test for chromosome damage and chemical toxicants that interfere with cell mitosis. Micronucleus is a kind of particle that exists outside the main nucleus in the cell, the size is equivalent to 1/20~1/5 of the cell diameter, round or almond-shaped, its staining is consistent with the nucleus, and one or more can appear in interphase cells. It is generally believed that the micronucleus is the genetic material that remains outside the nucleus in the late mitotic phase of the cell when the intracellular chromosomes are broken or the spindle filaments are affected. Therefore, the micronucleus test detects two genetic endpoints, altered chromosome integrity and altered chromosome segregation, induced by chemical toxicants or physical factors. Micronuclei can be found in a wide variety of cells, but are more difficult to distinguish from normal nuclear foliation and nuclear prominence in nucleated cells. Micronuclei are usually counted in PCE cells because erythrocytes can expel the primary nucleus several hours after the last separation prior to maturation, while still retaining micronuclei in PCE cells.
Materials and Instruments
Mouse Move 1. Test animals and treatment (1) Animal selection: generally used test animals for rats and mice. Mice are the most widely used and are required to weigh 18-20g and be 7-12 weeks old. The number of mice in each group is 10, half male and half female. (2) Poisoning route: According to the research purpose or the nature of the chemical poison under test, we can choose to use oral, percutaneous, respiratory tract and injection and other poisoning routes. But in principle, should be used as far as possible with the human body in contact with chemical poisons the same way. (3) The number of times of poisoning and sampling time: research has confirmed that chemical poisons need to accumulate in the target organ to a certain concentration before having a mutagenic effect. The peak time for the appearance of micronuclei induced by different chemical poisons varies. The range of fluctuation can reach 24-72 h. This requires the establishment of different sampling time points after exposure to chemical poisons. Considering the above two reasons, it is recommended to use the method of multiple poison staining. Among them, 4 times of poisoning is more convenient and reasonable. That is to say, the poison is dyed once a day for 4 consecutive days, and the sample is taken on the 5th day. In this way, sampling once can cover the 24-72h peak, and if the peak is delayed to 96h will not be missed. (4) Dose selection: the maximum dose of the tested chemical poison, in addition to being limited by the size of the solubility, should reach the maximum tolerance. Ye generally, there should be 3 to 5 or more dose groups, and the range of dose coverage should be more than 3 orders of magnitude. There should also be a positive control group and a negative control group. In the positive control group, cyclophosphamide (50-100 mg/kg) or mitomycin C (10 mg/kg) can be administered intraperitoneally once or twice. Equal volume of solvent was used in the negative control group. 2. Preparation and smear of bone marrow fluid After the test animals were last poisoned, they were executed by cervical dislocation or anesthesia according to the determined time, the limbs were fixed on the dissection board, the midline of the abdomen was wetted with hair, the thorax and abdomen were dissected, the sternum was removed, the blood was wiped off, the muscle was picked out, the epiphysis was cut off, the bone marrow was squeezed on the clean slide with a drop of calf serum using the small curved hemostat and the slides were pushed after mixing uniformly. Alternatively, after the animals were executed, the femurs of both legs were quickly removed with surgical scissors, the muscles were picked off, the blood stains and minced meat were washed away with saline, the epiphyses at both ends were cut off, calf serum was aspirated with a 2 ml syringe with a needle and inserted in the bone marrow cavity, the bone marrow was washed into the centrifugal tube, and the bone marrow was then blown into the bone marrow mass with a pipette to make it homogeneous, and then it was centrifuged for 10 min at 1,000 r/min, and the excess supernatant was discarded, leaving about 0.5 ml of the supernatant was discarded, leaving about 0.5 ml to be mixed with the precipitate, and then a drop was aspirated and placed on a clean slide with a dropper and pushed on the slide. Positive and negative control groups were processed at the same time according to the above method. 3. Fixation Put the pushed and dried bone marrow slides into the staining vat, fix them with methanol solution for 15min, and take them out to dry. Smears that cannot be stained in time should also be fixed and stored. 4. Staining The fixed and dried smears should be stained with freshly prepared Giemsa application solution. Giemsa application solution (Giemsa reserve solution l plus pH 6.8 phosphate buffer 9) staining 10 ~ 15min, then rinse off the staining solution on the slide, placed on the drying rack to dry. 5. Observation and counting Observe under low magnification first, choose the area with uniform distribution and good staining, then observe and count under oil microscope, PCE cells were gray-blue, and normal staining erythrocytes (NCE) were orange-yellow. Most of the micronuclei contained in the cells were round, with smooth and neat edges, and the chromophores were consistent with the nucleoplasm, which was purplish red or bluish purple. One or more micronuclei may be present in a single cell. The number of PCEs containing micronuclei was counted in 1,000 PCEs and the ratio of PCE to NCE was counted in 200 cells. Caveat Only micronuclei in PCE were counted in this test, and the micronucleus rate was expressed as a percentage in thousands. Each animal was a unit of observation. The mean values of micronuclei PCE were calculated separately for female and male animals in each group. The results can be combined when there is no obvious sex difference between female and male animals, otherwise the calculations should be performed separately; the normal PCE/NCE ratio is about 1 (normal range 0.6-1.2). If the ratio is ＜0.1, it means that PCE formation is severely inhibited, the dose of the tested chemical toxicant is too high, and the test result is unreliable. The incidence of micronuclei in the negative and positive control groups should be consistent with the results reported in the literature for the species and strains of animals used in the test or with historical data from the study. Common Problems The frequency distribution of the data obtained from the micronucleus test is inconclusive, and a variety of statistical methods (e.g., Poisson distribution, binomial distribution, X2 test, etc.) have been used to statistically analyze the results of the test. The key steps in this test are the production of good bone marrow smears and good quality staining. For more product details, please visit Aladdin Scientific website.
Methanol Glycerol Calf serum Saline Giemsa stock solution
Scalpels, surgical scissors, toothless forceps, small curved hemostatic forceps, tabletop centrifuge, graduated centrifuge tubes, glass crayons, glass staining cylinders, 2 microscopes with oil lenses, cell counters.