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

Inactive Publication Date: 2012-05-23
天津天诚新药评价有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Since the temperature will affect the drying time of the droplet, it will also affect the dispersion effect to a certain extent.
Due to the vast territory of our country, there are significant differences in climatic conditions throughout the year, and the temperature and humidity of laboratories in various places vary greatly. It is difficult to achieve suitable conditions for chromosome preparation and ensure the stability of the evaluation system only by relying on the external natural environment.

Method used

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  • Method for preparing metaphase specimen of mammalian cell chromosome
  • Method for preparing metaphase specimen of mammalian cell chromosome
  • Method for preparing metaphase specimen of mammalian cell chromosome

Examples

Experimental program
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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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Abstract

The invention provides a method for preparing a metaphase specimen of mammalian cell chromosome. The method comprises the following step of: dripping mammalian cell suspension onto a glass slide at the temperature of 20-30 DEG C and under the relative humidity of 45-50%. The metaphase chromosome obtained with the method is good in quality, shape and dispersion degree, and is a metaphase chromosome specimen easy for chromosome evaluation.

Description

Technical field [0001] The invention relates to a preparation method of mammalian cell chromosome specimens, in particular to a preparation method of mammalian cell chromosome metaphase specimens. Background technique [0002] In the development of new drugs, the evaluation of health products, and the research of radiation safety, the observation of chromosomal aberrations in mammalian cells is an important content in the evaluation of genetic toxicity. In the evaluation of chromosome aberrations, the preparation of chromosome metaphase specimens is a very critical link. The quality of chromosome dispersion is of great significance to the quality of the experiment, which in turn affects the reliability of the evaluation. Under-dispersed metaphase chromosomes are easy to cross and overlap, which makes it difficult to make accurate judgments when analyzing chromatids and chromosomes breaks, deletions, and interchanges. How to obtain metaphase chromosomes with good morphology has a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02
Inventor 申秀萍傅鹏张建军姜凌王建君
Owner 天津天诚新药评价有限公司
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