Micronucleus Detection Experiment
Micronucleus Detection Experiment
The MicronucleusTest was developed in the early 1970s as a rapid and simple method of detecting environmental carcinogens. Micronuclei are the remnants of chromosomal aberrations that can be observed when cells are in interphase, and manifest themselves with some regularity in the cytoplasm of lymphocytes from the terminal blood of cancer patients. Source of content: tissue culture and molecular cytology techniques. (Beijing Publishing House)
Operation method
Micronucleus Detection Experiment
Principle
Genotoxic or mutagenic factors acting on the chromatin, mitotic chromosomes and spindle of interphase cells can cause chromosomes to break into fragments or whole chromosomes to detach from the spindle and chromosome substrates to form backward isolated chromosomes, followed by the formation of an emblematic nucleus detached from the main nucleus at the end of prophase and in subsequent interphase cells. Thus micronuclei are essentially the form in which isolated aberrant chromosomes exist in interphase. Micronuclei can be detected either directly in vivo or using cultured cells.
Materials and Instruments
Venous Blood Serum Move 1. Method I Caveat 1. The micronucleus method is simple and easy to use, but is only suitable for the detection of factors that have an effect on the spindle and chromosomes during cell division. 2. It may not be very sensitive to substances of low toxicity; micronucleus detection has an error of about 30 %. Common Problems All cultured cells with the ability to divide and easy to obtain, as well as many other cells, can be used to display micronuclei. 1. Human peripheral blood lymphocyte culture For more product details, please visit Aladdin Scientific website.
Heparin Culture medium Eagle PHA Methanol Glacial acetic acid Giemsa Methyl cellulose Gelatin
Square bottles Centrifuge Sedimentation tubes
(1) Blood collection
Intravenous blood was collected 0.5 ml, anticoagulated with heparin, and cultured in small square vials with 5 ml of culture fluid;
Composition of culture solution is: Eagle's solution containing 20 % calf serum, plus PHA 40 μg/ml;
(2) Culture
(2) Incubate at 37 ℃ for 48 or 72 hours, and centrifuge at 1000 rpm for 8 minutes;
(3) Fixation
(3) Fixation: 3 L of 1 methanol/glacial acetic acid for 3 times, each time for 15 minutes, and centrifuge to precipitate the fixative;
(4) Preparation
(4) Preparation of frozen slide, natural drying; 10 % Giemsa (pH 6.8) staining for 15 min.
2. Method 2
(1) Blood collection
Heparin anticoagulated blood 0.5-0.6 ml, put in a 10 ml test tube, add half of the blood volume of 0.5 % methylcellulose, mix thoroughly;
(2) Culture
Place in a 37 ℃ incubator and incubate for 30 minutes, centrifuge at 2000 rpm for 10 minutes and remove the supernatant;
(3) Preparation
Make smears and stain with Wright's method.
3. Method 3
(1) Blood collection
Take 0.6 ml of blood from the ring finger by pricking with a three-pronged needle, immediately inject it into a blood sedimentation tube with an inner diameter of 3 mm and a length of 5 cm, and remove the fibrin by stirring with a small glass rod. tube, and remove fibrin by stirring with a small glass rod;
(2) Add gelatin
Add 3% gelatin (the amount of 1/3 or 1/2 of the blood volume), mix carefully, and place it in a 37 ℃ incubator. Natural sedimentation for 50 minutes;
(3) Aspirate the supernatant, centrifuge, smear the precipitate, dry naturally, stain with Wright's stain for 30 minutes, and then stain with Giemsa's stain for 2 minutes. Giemsa staining for 2 minutes.
It is very easy and effective in detecting mutagenic and carcinogenic substances in the environment, and is important in predicting harmful factors that may endanger the human body.
2. diploid cells
Tissue culture of artificial ploidy fibroblasts and other cells.
3. animal bone marrow cells
Mammalian bone marrow multi-stained erythrocytes: can be directly in vivo after the administration of the drug to do cell smears show micronuclei, is currently the most standard micronucleus indicator cells.
4. animal hepatocytes
Mammalian hepatocytes can metabolize and activate a variety of drugs and can be used to detect indirect carcinogens.
5. human oral epithelial cells
Human oral mucosa exfoliated cells: after making oral smear, direct staining shows micronuclei, simple and easy.
6. Plant cells
Onion root tip cells, duckweed, etc. for the detection of environmental mutagens, carcinogens, simple and effective.
1. Existence of temporal phase
Micronuclei must be in interphase cells with intact cytoplasm and round or oval shape.
2. Volume
The diameter is less than 1/5 of the primary nucleus.
3. Relationship to the primary nucleus
Completely detached from the primary nucleus.
4. Staining
Staining consistent with the primary nucleus.
5. Counting
Count 2000 cells per case under high magnification and express the results as a percentage of micronucleated cells per thousand.