Chromosome specimen preparation experiments
Chromosome specimen preparation experiments
Chromosomes are visible structures that appear during mitosis in eukaryotic cells, and only by obtaining chromosome specimens can karyotyping be performed to detect whether eukaryotic cells have abnormal chromosome numbers or structural aberrations.
Principle
The basic principle of chromosome specimen preparation is that human chromosome specimens are mainly taken from peripheral blood leukocytes, skin tissues, chorionic villus cells, amniotic fluid cells, bone marrow and so on. Usually, a small amount of peripheral venous blood, chorionic villus or amniotic fluid cells can be taken, short-term culture can be done to obtain a sufficient number of dividing cells, and the spindle filaments formed by microtubules can be depolymerized by colchicine treatment, so that the dividing cells will stop in the middle stage of the division, and then more middle stage chromosomes can be obtained for analysis after hypotonic and fixation treatment.
Operation method
Chromosome specimen preparation experiments
Principle
The basic principle of chromosome specimen preparation is that human chromosome specimens are mainly taken from peripheral blood leukocytes, skin tissues, chorionic villus cells, amniotic fluid cells, bone marrow and so on. Usually, a small amount of peripheral venous blood, chorionic villus or amniotic fluid cells can be taken, short-term culture can be done to obtain a sufficient number of dividing cells, and the spindle filaments formed by microtubules can be depolymerized by colchicine treatment, so that the dividing cells will stop in the middle stage of the division, and then more middle stage chromosomes can be obtained for analysis after hypotonic and fixation treatment.
Materials and Instruments
Consumables & Instruments: Move The basic process of chromosome specimen preparation experiments can be divided into the following steps: 1、On the outer flame of the alcohol lamp, use a disposable syringe to extract about 0.2 mL of heparin solution, put the sterilized paper sleeve of the syringe on the needle, pump the syringe so that the heparin is moistened up to the 5 mL mark of the syringe, and then discharge the remaining heparin. 2、After routine sterilization, collect about 5 mL of blood from peripheral vein of the elbow, and pump the syringe to make the blood and heparin mix well. 3、In the ultra-clean bench, add RPMI-1640 liquid medium (8 mL), calf serum (2 mL) and phytohaemagglutinin (0.7 mL) into the sterilized vials sequentially, and then add 30 drops of whole blood into each vial, and then shake horizontally to mix well. 4. Place the vials in a 37 ℃ constant temperature incubator and incubate for 70 h. (After 24 h of incubation, gently shake the vials to make the blood cells evenly suspended, and then continue to incubate). 5. Add colchicine to reach a final concentration of 0.2 μg/mL medium. Shake the bottle gently to mix the colchicine in the medium, and continue to incubate for 72 h. At the same time, place the 0.075 mol/L KCl hypotonic solution in a 37 ℃ thermostatic incubator for pre-warming. 6、Suspend the culture, remove the stopper, suck the culture solution with a nipple pipette, rinse the wall of the bottle repeatedly, so that the adherent cells are detached from the bottle wall, and then suck all the culture solution into 10 mL graduated centrifuge tubes. 7, leveling, 1000 r/min centrifugation 6~8 min. 8、Discard the supernatant, add 9 mL of 0.075 mol/L KCl hypotonic solution pre-warmed at 37 ℃, gently blow away the cell mass with a pipette, mix well, and then put it into 37 ℃ hypotonic treatment for 10~15 min. 9, add 1 mL of newly prepared fixative (3 parts methanol : 1 part glacial acetic acid), mix gently and centrifuge at 1000 r/min for 6 min. 10、Discard the supernatant, add 10 mL of freshly prepared fixative (3 parts methanol : 1 part glacial acetic acid), gently blow away the cell clusters to make a cell suspension, and then fix it for 30 min at room temperature. 11. Centrifuge the cells at 1500 r/min for 6 min. 12、Discard the supernatant and repeat the fixation once. 13、Discard the supernatant, add a few drops of newly prepared fixative according to the number of cells, and gently blow the cells into suspension. 14: Pipette a small amount of cell suspension, put 2~3 drops at a height of about 20 cm on a slide pre-cooled at 4 ℃, blow it away and air dry. 15: Take 3 drops of Giemsa's original solution and add 10 drops of phosphate buffer (pH 6.8), mix well and drop on the slide specimen, stain for 6 minutes. 16、Rinse the slide gently with running water to wash out the staining solution and air dry. 17、Browse the whole chromosome slide specimen with low magnification first, and then observe the number and dispersion of split phase of chromosome specimen under high magnification and oil microscope respectively.
Alcohol lamp, blood collection equipment, culture bottles, ultra-clean bench
Thermostatic incubator, thermostatic water bath, 10 mL graduated centrifuge tubes, nipple pipettes, low-speed centrifuge
4 ℃ pre-cooled slides, hair dryer, pallet balance, microscope, human peripheral venous blood, chorionic villus cells, amniotic fluid cells
Chorionic villus cells, amniotic fluid cells
Reagents:
RPMI-1640 liquid medium, HamF10 culture medium,
calf serum, heparin (500 U/mL), phytohaemagglutinin (PHA)
Phytohemagglutinin (PHA)
Colchicine (10 μg/mL)
0.075 mol/L KCl hypotonic solution, 0.4% KCl
0.4% sodium citrate, 70% acetic acid
0.85% physiological saline
Fixative (3 parts methanol : 1 part glacial acetic acid, ready to use)
Giemsa stock solution, cedar oil, xylene
1. Enter the uterine cavity through the cervix and aspirate about 5 mL of chorionic villi.
2. Rinse the aspirated villi repeatedly with 0.85% saline to remove blood stains.
3. Place the villi in a centrifuge tube containing 5 mL of serum-free RPMI-1640 culture medium, add colchicine (so that the final concentration is 1 μg/mL of medium), mix well, and then incubate for 18-20 min at 37 ℃.
4. Add 8 mL of hypotonic solution mixed with 0.4% KCl and 0.4% sodium citrate into the centrifuge tube, and incubate at 37 ℃ for 10~12 min.
5. Add 2 ml of freshly prepared fixative (3 parts methanol : 1 part glacial acetic acid), and fix for 3 min at room temperature.
6: Absorb all the liquid as much as possible, add 1 mL of 70% acetic acid, leave to dissociate the cells for 1~2 min, immediately add 2 mL of methanol and mix well.
7. Slowly add 5 mL of freshly prepared fixative, mix well and continue fixation at room temperature for 30 min.
8. Centrifuge at 1200 r/min for 8 min, discard the supernatant, add 5 mL of freshly prepared fixative, and repeat fixation for 30 min.
9、Blow the cell suspension gently with a pipette and pick off the villous scaffold. 1200 r/min centrifugation for 8 min.
10、Discard the supernatant, add a few drops of freshly prepared fixative to make cell suspension, and then drop the slice.
11, the subsequent staining microscopy steps with 1.
Third, the culture of amniotic fluid cells and chromosome specimen preparation1, aseptically absorb 10 mL of amniotic fluid, placed in a 10 mL centrifuge tube, centrifuged at 1200 r/min for 5 minutes.
2、Discard the supernatant, keep 0.5 mL of amniotic fluid/cell layer, blow evenly with a pipette, inoculate it into 5 mL of HamF10 culture medium, and incubate it in 37 ℃ for 5-7 d. 4 hours before the termination of culture, the cells should be cultured for 5-7 d. The culture should be incubated for 4 hours before the termination of culture.
3. 4 h before the termination of culture, add colchicine to make the final concentration of 0.25 μg/mL, and continue the culture.
4、Rinse the wall of the bottle repeatedly with a pipette, so that the adherent cells were fully dislodged, and then the cell suspension was sucked into a centrifuge tube.
5、Centrifuge at 1200 r/min for 8 min, and discard the supernatant.
6、Add 3~4 drops of hypotonic solution mixed with 0.4% KCl and 0.4% sodium citrate pre-warmed at 37 ℃, mix well and continue to add hypotonic solution to 1 mL, incubate at 37 ℃ for 5 min.
7. Add several drops of newly prepared fixative (3 parts methanol : 1 part glacial acetic acid) and mix gently for pre-fixation.
8、Centrifuge at 1200 r/min for 8 min, and discard the supernatant.
9. Add 4 mL of freshly prepared fixative and fix for 40 min at room temperature.
10, 1200 r/min centrifugation 8 min, discard the supernatant.
11. Repeat fixation with 2 mL of freshly prepared fixative.
12. Centrifuge at 1000 r/min for 10 min and discard the supernatant.
13, add 2~3 drops of newly prepared fixative to make cell suspension and drop the slice.
14, later staining microscopy steps are the same as 1.
Caveat
1. When taking blood for inoculation culture, pay attention to the dosage of heparin, too much heparin may lead to hemolysis and inhibit the transformation and division of lymphocytes; too little heparin may lead to coagulation or the appearance of membrane structure formed by fibrin in the cultures (the membrane can be removed in aseptic conditions).
2. In the semi-microculture method, the inoculum is whole blood, which contains more red blood cells and is prone to red blood cell coagulation; after 24 hours of inoculation and culture, the bottle must be gently shaken to disperse the cells so as not to affect the culture effect.
3. For chromosome specimen preparation, the most critical factors affecting the quality of specimen are the amount and time of colchicine and the treatment time of hypotonicity.In the culture of peripheral blood lymphocytes, their division peaks at 70~72 h. Therefore, the time of adding colchicine can be around 68~70 h of culture, and the final concentration is 0.2 μg/mL.If the quality of colchicine is faulty or the concentration of colchicine is too low, the treatment time is insufficient or inappropriate, the result will be fewer division phases; if the concentration of colchicine is too high or the treatment time is too long, the chromosomes will be too shortened, which makes it difficult to carry out the karyotype analysis.For hypotonic time, if the processing time is too long, the cell membrane tends to rupture prematurely, resulting in the loss of split cells or chromosome loss; if the processing time is insufficient, the cell expansion is not enough, the chromosome dispersion is not good, it is difficult to carry out chromosome counting analysis.
4. Fixing solution must be prepared now.
5. Slides must be degreased beforehand, and should be stored at 4 ℃ or pre-cooled with ice-water mixture before preparation.
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