Chromosome preparation experiments on cultured peripheral blood cells
Chromosome preparation experiments on cultured peripheral blood cells
Although chromosomes can be prepared from other cells, human peripheral blood leukocytes are the easiest to synchronize, allowing high-resolution analysis of their chromosomes.
Operation method
Peripheral blood culture and mid-division cell harvesting assay
Materials and Instruments
Heparinized whole blood is obtained with Vacutainer (BectonDickinson) or a syringe containing preservative-free heparinized Na. （Heparinized whole blood is obtained using Vacutainer (BectonDickinson) or syringes filled with preservative-free heparin na.ml (25U ml). Move Basic protocols Peripheral blood culture and mid-division cell harvesting 1. Collect peripheral blood by venipuncture using a Vacutainer containing sodium heparin or a syringe containing 25U/ml blood preservative-free sodium heparin (do not use other anticoagulants). Start culture as soon as possible (recommended) or store at 4°C for no more than 4 d. Transport at room temperature. 2. Using a TB syringe with a 21 G needle, inoculate 0.25 ml of whole blood into a sterile 15 ml polypropylene centrifuge tube containing 5 ml of complete RPMI with 10% FBS and gentamicin. Add 0.2 ml of whole blood for neonates <3 weeks of age, not to exceed 0.5 ml in any one tube, and 0.05 ml of redissolved 100XPHA. Make 3 to 5 spread slides (or more spread slides) per culture tube. 3a. Standard samples: incubate tubes at an angle of 45° (- a certain angle facilitates air exchange and prevents accumulation of sediment) for 2 to 4 d (preferably 3 d). 3b. Neonates: harvest directly as described or after 1~2d incubation (2d is optimal). 3c. Elderly: lymphocytes in this age group are slower to respond to PHA and are therefore harvested after 3-4d of culture. 4. Optional: For longer chromosomes and more mitotic cells, apply the following method (Figure 4.1.1) to obtain better synchronized cells. One day before harvest, add 0.05 ml of 10 mmol/L methotrexate (final concentration 10-7mol/L) to block DNA replication. Continue incubation for 16~18 h (not more than 18 h). On the second day, add 0.05 ml of lmmol/L deoxythymidine nucleoside (final concentration 10-5mol/L) to unblock methotrexate. Incubation was continued for approximately 4 h (time was the critical factor). 5. After 3-4 d of incubation (step 3), or synchronization (step 4), directly add 25 ul of lOug/ml colchicine (final concentration of O.OSug/ml), initiate the harvest, and incubate for 30 min. Centrifuge at 180 g for 8 min at room temperature and discard the supernatant. 6. Add 6 ml of 75 mmol/L Ka solution at room temperature and gently resuspend the cells. Allow to stand at room temperature for 15 min. Adjusting the volume of KCl according to the volume of precipitate to achieve optimal conditions is more effective than prolonging the time. 7. Add 10-12 drops of fixative using a pasteurized tube and mix well. Centrifuge as in step 5. 8. Reserve 0.5 ml of supernatant and gently aspirate using a pasteurized tube to resuspend the brown mass. Avoid aspirating so much that it sticks to the glass tube. Do not press the tip of the pipette firmly against the bottom of the tube. Add 1 ml of fixative and mix gently and immediately, adjust volume to 5 ml with fixative and mix well. Centrifuge as in step 5. 9. Discard supernatant, resuspend precipitate with 5 ml of fixative and centrifuge as in step 5. 10. Dispose of the supernatant and resuspend the precipitate in an appropriate volume of fixative to a milk-like suspension. Allow to stand for 30 mm at room temperature or overnight at 4°C (longer fixation times are better for chromosome dispersion). 11. Produce and analyze the dispersed chromosomes (support protocol). This method is suitable for cells cultured by a variety of methods: peripheral blood, bone marrow (Unit 10.1), peritoneal fluid, pleural fluid, amniotic fluid (Unit 8.2), and culture flask-harvested cells (Units 8.1, 8.3, and 10.2), hybridized or radial hybridized somatic cells (Unit 3.1), lymphoblastoid cell lines, and non-human hybridoma cells. In summary, the method is suitable for the preparation of chromosome slides from any cultured, fixed mitotic cell suspension. 1. Remove the slide from the methanol and wipe it with a cotton free flannel (it is important that the slide is clean). Re-immerse the slide in methanol and rinse repeatedly with deionized water until the methanol is washed out and a uniform film of water remains on the surface of the slide. 2. Hold the frosted end of the slide with your thumb and forefinger, tilt the slide so that its long side is parallel to the work surface, and touch the lower long side of the slide with blotting paper to soak up the excess water on the slide. Tilt the slide at an angle of 30° to the work surface and keep the lower long edge of the slide in contact with the blotting paper, with the watery side up (Fig. 4.1.2). 3. Hold the pasteurized tube horizontally 1 to 2 in above the slide and add 3 drops of cell suspension continuously and evenly, moving in the direction of the frosted end. Each drop occupies an average of 1/3 of the width of the slide. Drops on an inclined slide are dispersed as they touch the slide. If the area of cell dispersion is concentrated at the bottom of the droplet and there are fewer cells around the droplet, the angle between the slide and the bench should be reduced during the drop (<30°). 4. Pipette off the excess fixative, tilt the slide at 30° as in step 2, and add drops of freshly prepared fixative, drop by drop, using a pasteurized tube. Starting from the elevated unfrosted end and moving towards the frosted end, drop on the elevated edge of the slide to form a uniform front of fixative. 5. Blot the lower long edge of the slide again for moisture and wipe the back of the slide dry. Tilt the frosted end of the slide upward by 30 degrees, with the cell side up, and allow to dry in the air for adequate dispersion. If the cells are not well dispersed around the periphery of the slide, see Table 4.1.1 (e.g., adjust the temperature to 20?22°C and relative humidity to 50%). 6. Look for well-dispersed chromosomes with good morphology under a phase contrast microscope. Discreetly select slides with even and uniform chromosome distribution, moderate density, and no overlapping chromosomes. To facilitate the acquisition of ideal chromosome bands or hybridization signals, ideal chromosomes should be flat, well-banded, and consistently colored. 7. Store slides in a clean, dry, dark place at room temperature (short-term) or freeze at 70°C (long-term). For more product details, please visit Aladdin Scientific website.
Complete RPMI 10% FBS (fetal bovine serum) medium 100 X phytohemagglutinin Amethopterin Deoxythymidine Nucleoside Colchicine Potassium chloride Fixative
Sterile conical polypropylene centrifuge TB syringe
