Source: Laboratory Course in Genetics
Operation method
basic program
Principle
Peripheral blood is one of the most important materials for the preparation of animal chromosome specimens, but usually there is no schizogony in the peripheral blood of mammals, and schizogony can only be seen occasionally in the peripheral blood of other animals such as amphibians. Almost all small lymphocytes in peripheral blood are in the G1 or G0 phase, and when phytohemagglutinin (PHA) was added to the culture medium of artificial in vitro culture, small lymphocytes were stimulated to transform into lymphoblasts, which then entered into the dividing phase. Thus, after a short period of culture, a large number of mid-mitotic cells could be obtained by treatment with colchicine for the preparation of chromosome specimens.
Materials and Instruments
Human peripheral blood lymphocytes Move 1.Culture medium: In the ultra-clean bench, take 40ml of "1640" culture medium, 10ml of serum with a measuring cylinder, take 0.3ml of heparin, 0.4ml of PHA, 0.3ml of double antiseptic with a 1ml syringe (the final concentration of each is 140 units/ml), mix well, adjust the pH value to 7.2 with 5% NaHCO3, put 5ml of 5ml pipette into 20ml culture bottle, 5ml of each vial, cover the stopper tightly. After mixing, the pH was adjusted to 7.2 with 5% NaHCO3. 5ml pipettes were used to dispense into 20ml culture vials, 5ml per vial, capped tightly with stoppers and sealed with adhesive tape. If not used immediately can be placed in 0 ℃ storage, before using 37 ℃ water bath treatment 10min can be. 2. Blood collection: wash the blood donor's hand with soap, then wipe the fingertip with 2% iodine and 75% alcohol to disinfect it, after the alcohol evaporates and dries completely, prick the fingertip with a sterile blood collection needle or a three-pronged needle, use a sterile pipette to suck 0.3-0.4ml of blood into the culture bottle, shake gently to make the blood mixed with the culture medium can be cultured (or use a 2 ml syringe with a No. 7 needle, suck a little heparin solution to moisten the needle, and then collect blood from the elbow vein for 1～1ml. Collect 1~2ml of blood from the elbow vein, and inoculate each culture bottle with about 0.3ml of whole blood.) 3. Cultivation: The culture bottle with blood cells was placed in 37℃ incubator for 68～72h. 4. Colchicine treatment: add colchicine (2μg/ml) 6～10h before the termination of culture, add 3～4 drops with a No.5 needle, so that the final concentration of 0.01～0.02g/ml, and treat for 2～4h. 5. hypotonic treatment: take out the culture bottle from the incubator, suck off the supernatant with a pipette, the culture was deposited at the bottom of the bottle, add about 6.5 ml of 0.075 mol/L KCl solution preheated at 37 ℃, and then put it into the hypotonic treatment at 37 ℃ for 20 min to make the red blood cells fragmented and the leukocytes swollen. 6. Centrifugation: centrifuge at 1000r/min for 5min, use a pipette to suck off all the supernatant and the transparent part of the upper layer of precipitation (erythrocyte fragments), and collect the leukocytes. 7. Fixation: slowly add 5-6ml of methanol-glacial acetic acid (3:1) fixing solution along the wall of the tube, blow evenly with a pipette and fix it for 20min. centrifuge at 1000r/min for 5min and discard the supernatant. 8. Preparation: Depending on the number of cells at the bottom of the centrifuge tube, add a small amount of fresh fixative and blow it into a homogeneous suspension. Take a slide pre-cooled in clean ice water, slightly tilted, with a pipette to suck a drop of the above cell suspension, in the appropriate height above the slide drop on the slide, immediately blowing blowing the cells on the slide, air-drying (the resulting preparation can be retained for the banding experiment). 9. Staining: After the slides were fully dried, they were stained with phosphate buffer at pH 6.8 by mixing 1 part of Giemsa stock solution and 9 parts of phosphate buffer. The material side up, cover the slide with the staining solution, staining for 10~15min, wash away the excess staining solution with running water, and then absorb the excess water with water-washing paper, then dry it for microscopic examination. Under the low and medium magnification microscope, search for the split phase with appropriate dispersion, no overlapping of chromosomes, moderate concentration and clear morphology, and observe the number and morphology of chromosomes under the oil microscope. A representative split phase was selected and micrographically photographed with a digital imaging system for karyotyping. Caveat 1. The fresher the inoculated blood sample, the better, preferably within 24h after blood collection for culture. If it is not possible to culture immediately, it should be stored at 4℃ to avoid keeping it for too long, which may affect the cell viability. 2. In addition to the potency of PHA, the culture temperature and pH of the culture medium are also very important for the success or failure of the culture. The optimal temperature for human peripheral blood lymphocyte culture is 37±0.5℃, and the optimal pH value is 7.2-7. 4. 3. In the process of culture, if the blood samples are found to be agglutinated, the culture bottle can be gently shaken to disperse the clots, and then put back to 37℃ for culture. 4. This experiment used human peripheral blood microculture method for human chromosome preparation, other animals can use the same method, just according to different objects to make appropriate changes. 5. If the cells are not swollen enough, the cell membrane is not ruptured, or the chromosomes are not dispersed enough, the hypotonic time can be extended appropriately. For more product details, please visit Aladdin Scientific website.
RPMI "1640" medium Calf serum Heparin saline solution (500 units ml) 5% NaHCO3 Colchicine (4 μg ml) PHA Dual Antibody (Penicillin 10,000 units ml, Streptomycin 10,000 units ml)
Ultra-clean bench Constant-temperature incubator Constant-temperature water bath Centrifuge 20 ml culture flask Syringes and needles Scissors and tweezers Beakers and measuring cylinders