Experiments on the preparation of chromosome smears in the mid-division of chorionic specimens
Experiments on the preparation of chromosome smears in the mid-division of chorionic specimens
The interstitial center of the villi includes a range of cellular components-fibroblasts, endothelial cells, and macrophages-as well as a large amount of intercellular matrix. Fibroblasts in the center of the villi are capable of rapid proliferation in vitro and are suitable material for cytogenetic analysis.
Operation method
Experiments on the preparation of chromosome smears at the mid-chromosome stage of chorionic division by the culture method
Materials and Instruments
Chorionic samples Move basic program 1. Carefully separate the villi from the maternal metamorphosis under an inverted phase contrast microscope using sterilized Watt's fine forceps. Transfer the villi to HBSS for washing and replace the HBSS until the villi are clean. Divide the villi into two parts, one for culture (step 2) and the rest for direct preparation (alternative, step 1). 2. Transfer 4-10 mg of finely chopped and washed villi into a 4.5 ml sterilized cold test tube containing 4 ml of trypsin/EDTA/HBSS solution. Incubate on a shaker tube mixer at 37°C for 60 min. Centrifuge at 800 g for 5 min at room temperature. 3. Gently aspirate the trypsin solution. Add 1.5 to 2 ml of collagenase solution and incubate for 30 min at 37°C in a shaker-tube mixer. 4. Centrifuge at 800 g for 5 min at room temperature and gently aspirate off the collagenase solution, leaving about 0.5 ml in the test tube. 5. Using a Pasteur pipette, gently blow the villi and the remaining collagenase solution 10 times to break the villi into a single cell suspension. Add 3ml of pre-warmed chang in situ medium to the suspension. 6. Place 0.5 ml of single cell suspension on the surface of a flame-sterilized coverslip in 2-6 labeled 35 mm plastic bacterial culture dishes. If the cell suspension on the coverslip is too thick (viewed under an inverted microscope), dilute it with chang in situ medium. Incubate for 24 h. 7. Add 2 ml of Chang's In Situ Medium to each dish and incubate until the cells are attached to the coverslip (2~3d). 8. After the cells were attached to the coverslips, aspirate the medium, add 2ml of fresh Chang's in situ medium, and continue to incubate. The best results are obtained from the mesenchymal center alone. If . There is excessive debris or contamination of parent cells (suspension cells), the coverslip must be carefully transferred into another in situ medium containing 2 ml Chang. Nest-like clones of fibroblasts will appear after 1~2d and the coverslips will be ready for harvesting after 5~7d of flat culture, depending on cell growth and mitotic activity (e.g., number and size of clones and number of dividing cells). 9. Add 10 ul of colchicine to each dish and incubate for lh. Remove the medium from the dish and add 3 ml of pre-warmed aconite. Incubate for 20 min. 10. Carefully add 2 ml of freshly prepared fixative at 4°C to the dish and leave for 2 min at room temperature. 11. Aspirate off the sodium citrate/fixative solution and reapply 2 ml of freshly prepared fixative at 4°C. Allow to stand for 20 min. Leave for 20 min. Repeat the fixation twice, the first time for 20 min and the second time for 10 min. 12. Pipette off the fixative, remove excess liquid and dry the coverslip immediately. Expose the coverslip surface to hot air for 1-3 s. Immediately place the coverslip surface in a gentle stream of air from the air pump. Observe the coverslip surface if necessary to ensure uniform drying and to adjust the airflow. Proper drying of the coverslip is critical to the success of the entire experiment. 13. Observe the coverslip under an inverted microscope to determine the degree of chromosome dispersion. If necessary, adjust the drying procedure to obtain a better split phase. 14. Stain the chromosomes by trypsin-Giemsa banding. 15. Photograph and analyze 7~10 intermediate schizogony phases, and photograph and karyotype 2 intermediate schizogony phases (Appendix 3K). Mid-division phase smears can be obtained from trophoblast cells (Langerhans cells of the cellular trophoblast) spontaneously dividing by mitosis or from trophoblasts that have been cultured for a limited period of time (e.g., just a few hours). 1. Place the remaining fluff not used in the culture method into a dish containing 3 ml of pre-warmed 37°C complete RPMI/20% FBS with 1% gentamicin. Incubate for more than 3 h (never less than 3 h). 2. Synchronize the culture by adding 0.1 ml of FUdR (final concentration 3. 3X10-7mol/L ) at a concentration of 0.0 mmol/L. Incubate for 15~17 h. 3. Add lmmol/L thymidine (final concentration 3. 3X10-5mol/L ) 0.1 ml. Incubate for 5 h. (If there is no adverse effect on the harvest rate in the middle stage of division, the incubation time in thymidine can vary from 3 to 8 h). 4. Add colchicine to a final concentration of 0.5ug/ml and incubate for 2 h. Remove the petri dish from the incubator and gently aspirate the medium with a pasteurized pipette. 5. Slowly add 3 ml of 1% sodium citrate at 37°C and incubate for 20 min. carefully add 0.5 ml of 4°C fixative to terminate the hypotonic effect of sodium citrate. 6. Aspirate off the sodium citrate/fixative solution and add 2 ml 4°C freshly prepared fixative. Shake the Petri dish to rinse the villi and aspirate the fixative. Add 2 ml of fixative and place in refrigerator overnight (20 min if results are needed within 24 h). 7. Remove the fixative and leave at room temperature for 1~2 min to allow the residual fixative to evaporate. 8. Add 100~200 ul of 60% glacial acetic acid. Observe the release of cells from the villi under an inverted phase contrast microscope. Shake gently. Slides can be made when the release of cells is very obvious (individual cells are free from the tissue). Check the number of individual floating cells. 9a. Place a labeled slide on the surface of a slide warmer that has reached 45°C. Place 100ul of the mixed cell suspension on the slide and gently spread the suspension over the entire slide with the applicator. 10a. By moving the slide slowly with the other hand without using the applicator, the applicator can disperse the cell suspension along the axis of the slide to cover 50%~60% of the center of the slide. Repeat 3~5 times, 30~60s for each round trip (3~5 min in total), so that the cell suspension can be fully spread and dried. 11a. Remove the slide from the slide warmer and blot the residual liquid from the edge of the slide. Observe the dispersion of the split phase under an inverted microscope. Allow the slides to incubate overnight at 65°C or for 10 min at 90°C before staining (step 12). 9b. Set the temperature of the slide maker to 45°C and place the labeled slides in the slide holder. Place 100ul of mixed cell suspension on each slide and lower the comb (the machine can produce 6 slides at a time). 10b. Run the slicer for 4-5 min. remove the slides and blot the residual liquid from the edges of the slides. Observe the dispersion of the split phase under a phase contrast microscope. Allow the slides to incubate overnight at 65°C or lOmin at 90°C before staining. Perform step 12. 12. Stain the chromosomes by Trypsin-Giemsa Banding (Module 4.3). Analyze the results of 7 to 10 midsegmentation images. For more product details, please visit Aladdin Scientific website.
HBSS trypsin EDTA HBSS solution Collagenase solution Complete medium Colchicine amine Sodium citrate Formaldehyde Glacial acetic acid
Sterile Watchmaker Fine Forceps Cold Test Tubes Shaker Tube Mixer Sorvall GLC-2B Centrifugal Equipment HL-4 Rotor and 6 15ml Centrifuge Tubes Plastic Petri Dish Inverted Microscope or Dissecting Microscope Hot Air Humidifier Air Pumps