Method for preparing metaphase specimen of mammalian cell chromosome
A technology of mammals and chromosomes, applied in the field of preparation of mammalian cell chromosome metaphase specimens, can solve problems such as difficulty in ensuring the stability of the evaluation system, large differences in laboratory temperature and humidity, and difficulty in achieving suitable conditions for chromosomes
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Embodiment 1
[0045] Mammalian cells are CHL cells (Chinese hamster lung fibroblasts, purchased from Shanghai Institute of Cells) as an example. CHL cell culture conditions: RPMI1640 culture medium containing 10-15% (10-15ml serum / 100ml RPMI1640 culture medium) fetal bovine serum, 37℃, 5% CO 2 Culture in an incubator.
[0046] Resuscitate CHL cells, digest the passage cells with 0.25% (g / 100ml) trypsin aqueous solution to prepare 3~5×10 4 Cells / mL cell suspension, add 2mL cell suspension and 8mL fresh medium to each 50mL culture flask, at 37℃, 5% CO 2 Cultivate in the incubator for 2 days, replace the culture medium, and continue the culture. Cultivate for 21 hours, add 0.2 mL of colchicine (concentration 15 μg / mL), culture for 24 hours, discard the medium, and harvest the cells.
[0047] After digesting the cells with 0.25% (g / 100ml) trypsin aqueous solution, transfer to a 15mL centrifuge tube, centrifuge at 1000rpm for 10 minutes, remove the supernatant, add 5mL of 0.075mol / L KCl aqueous solut...
Embodiment 2
[0051] Mammalian cells take human peripheral blood lymphocytes as an example. CHL cell culture conditions: RPMI1640 culture medium containing 10-20% (10-20ml serum / 100ml RPMI1640 culture medium) fetal bovine serum, 37℃, 5% CO 2 Culture in an incubator.
[0052] Take venous blood from healthy volunteers and separate lymphocytes from anticoagulation. Add 1 mL of lymphocyte suspension and 9 mL of fresh medium to each 50 mL culture flask. At 37°C, 5% CO 2 Cultured in an incubator for 64 hours, added 0.2 mL of colchicine (concentration 15 μg / mL), cultured to 68 hours, discarded the medium, and harvested the cells.
[0053] The cell culture solution was transferred to a 15 mL centrifuge tube, centrifuged at 1000 rpm for 10 minutes, the supernatant was removed, and 5 mL of 0.075 mol / L KCl aqueous solution was added, and hypotonic for 20 minutes (37° C.). Add 0.5 mL of fixative (volume ratio: methanol: glacial acetic acid=3:1) to each tube, perform pre-fixation, centrifuge at 1000 rpm for ...
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