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Method for rapidly preparing prematurely condensed chromosomes of human peripheral blood lymphocytes

A technology of lymphocytes and human peripheral blood, applied in the field of cytogenetics, can solve the problems of premature agglutination of chromosomes and other problems, and achieve the effect of shortened culture time, short time consumption and fast access

Inactive Publication Date: 2014-05-21
NAT INST FOR RADIOLOGICAL PROTECTION & NUCLEAR SAFETY CHINESE CENT FOR DISEASE CONTROL & PREVENTION
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to establish a method for rapidly preparing prematurity agglutination chromosomes of human peripheral blood lymphocytes to solve the problem of time-consuming preparation of prematurity agglutination chromosomes of human peripheral blood lymphocytes in the prior art

Method used

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  • Method for rapidly preparing prematurely condensed chromosomes of human peripheral blood lymphocytes
  • Method for rapidly preparing prematurely condensed chromosomes of human peripheral blood lymphocytes
  • Method for rapidly preparing prematurely condensed chromosomes of human peripheral blood lymphocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] (1), collect 3ml of blood samples from normal volunteers, add PHA (Guangzhou Pharmaceutical Industry Research Institute) to the blood samples to a final concentration of 80 μg / ml, mix well and place in a place containing 5% CO 2 Stand in a 37°C constant temperature incubator for 1 hour;

[0020] (2) After the blood sample was left to stand in the above-mentioned 37°C constant temperature incubator for 1 hour, it was centrifuged at 300rpm for 3 minutes, and 0.5ml of blood lymphocytes at the middle layer was taken and placed in a medium containing 3ml of 250μg / ml colchicine (sigma company), 10mmol / L ATP (sigma company), 5% fetal bovine serum, 500nmolCalyculinA (sigma company) and 0.2mg / ml CDK1 / CyclinB (cyclin-dependent kinase 1 / cyclin B, Invitrogen company) mixed culture medium , mix well and place in a constant temperature incubator at 37°C for 3 hours;

[0021] (3) Lymphocytes were centrifuged at 1000rpm for 10 minutes after being mixed and cultured for 3 hours, and 5m...

Embodiment 2

[0025] (1), collect 3ml of blood samples from normal volunteers, add PHA (Guangzhou Pharmaceutical Industry Research Institute) to the blood samples to a final concentration of 100 μg / ml, mix well and place in a place containing 5% CO 2 Stand in a 37°C constant temperature incubator for 1 hour;

[0026] (2) After the blood sample was left to stand in the above-mentioned 37°C constant temperature incubator for 1 hour, after being centrifuged at 400rpm for 3 minutes, 0.8ml of lymphocytes at the middle layer junction was collected and placed in a solution containing 3ml of 250μg / ml colchicine, 10mmol / L ATP, 5% fetal bovine serum, 500nmol CalyculinA and 0.2mg / ml CDK1 / CyclinB mixed culture medium, mix well and place in a constant temperature incubator at 37°C for 6 hours;

[0027] (3) Lymphocytes were centrifuged at 1000rpm for 10 minutes after mixed culture for 6 hours, and 5ml of 0.075mol KCl hypotonic solution was added in a solution containing 5% CO 2 Hypotonicity for 20 minut...

Embodiment 3

[0031](1) Collect 5ml of blood samples from normal volunteers, add PHA (Guangzhou Institute of Pharmaceutical Industry) to the blood samples to a final concentration of 100μg / ml, mix well and place in 5% CO 2 Stand in a 37°C constant temperature incubator for 90 minutes;

[0032] (2) After the blood sample was left to stand in the above-mentioned 37°C constant temperature incubator for 90 minutes, it was centrifuged at 300rpm for 3 minutes, and 0.5ml of lymphocytes at the middle boundary was taken and placed in a medium containing 3ml of 250μg / ml colchicine, 10mmol / L ATP, 5% Fetal bovine serum, 500nmol CalyculinA and 0.2mg / ml CDK1 / CyclinB mixed culture medium, mix well and place in a constant temperature incubator at 37°C for 12 hours;

[0033] (3) Lymphocytes were centrifuged at 800rpm for 10 minutes after being mixed and cultured for 12 hours, and 4ml of 0.075mol KCl hypotonic solution was added in a solution containing 5% CO 2 Hypotonicity for 20 minutes at 37°C;

[0034]...

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Abstract

The invention relates to a method for rapidly preparing prematurely condensed chromosomes of human peripheral blood lymphocytes. The method mainly comprises the following steps: 1, processing a blood sample by phytohemagglutinin, allowing the processed blood sample to stand in a constant temperature incubator with the temperature of 37DEG C for 45-90min; 2, culturing lymphocytes in the phytohemagglutinin processed blood sample in a mixed culturing liquid containing colchicine, ATP, 5% fetal calf serum, CalyculinA and CDK1 / CyclinB at 37DEG C for 3-12h; 3, carrying out hypoosmotic treatment of the cultured lymphocytes by using 0.075mol of KCl for 10-20min; and 4, adding an immobile liquid into a hypoosmotic liquid containing the hypoosmotic lymphocytes to realize first-time immobilization, adding the immobile liquid to realize second-time immobilization, and suspending the obtained lymphocytes in the immobile liquid, wherein each of the first-time immobilization and the second-time immobilization is carried out for 10-15min. The preparation method provided by the invention takes a substantially shorter time of 3-12h than routine preparation methods of the prematurely condensed chromosomes.

Description

technical field [0001] The invention belongs to the field of cytogenetics, and in particular relates to a method for preparing prematurity agglutinated chromosomes of human peripheral blood lymphocytes. Background technique [0002] The most classic concept of Premature Condensed Chromosome (PCC) refers to the fusion of cells in the division phase (M phase) with cells in other phases of the cell cycle, and the chromatin of cells in other phases is packaged into chromosomes in advance. Due to the different DNA replication states of G1, S, and G2, the prematurity and condensed chromosomes have different shapes. For example, the chromosomes in the G1 phase fused with the M phase cells are single-threaded, the S phase is powdery, and the G2 phase chromosomes are double-threaded. [0003] Precocious condensed chromosome PCC has received extensive attention and recognition as a method for estimating radiation dose. Premature condensed chromosomes have strong advantages over conve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0781C12N5/0783
Inventor 刘青杰高玲陆雪陈德清
Owner NAT INST FOR RADIOLOGICAL PROTECTION & NUCLEAR SAFETY CHINESE CENT FOR DISEASE CONTROL & PREVENTION
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