Genetic disease refers to a disease caused by a change in genetic material or controlled by a disease-causing gene. At present, the diagnosis of genetic diseases mainly adopts the method of cytogenetics, namely karyotype analysis, which is of great significance in the diagnosis and screening of genetic diseases. The key to karyotyping is to culture qualified lymphocytes. The traditional lymphocyte culture medium uses a sodium bicarbonate buffer system, which is easily affected by carbon dioxide in the environment. As the storage time is prolonged, the pH value is increased, which is not conducive to the preservation of the medium. To overcome the above technical problems, the researchers used 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) to develop a more stable pH medium.
The lymphocyte medium is composed of 1640 basal medium, phytohemagglutinin (PHA, 100 mg/L), calf serum (20% by volume), and sodium glycerophosphate/HEPES Double buffer system (5~50mM). The sodium glycerophosphate/HEPES double buffer system consists of phosphate buffer (Na2HPO4 and KH2PO4), sodium glycerophosphate, and HEPE). The specific components and concentrations are shown in Table 1 below.
Component |
concentration |
Basic medium |
1640 medium |
PHA |
100mg/L |
Sodium glycerophosphate |
3g/L |
HEPES |
3g/L |
Na2HPO4 |
2g/L |
KH2PO4 |
1g/L |
Calf serum |
20% |
Take the components of the above medium, add to the ultrapure water, so that the components are fully dissolved; add ultrapure water to volume, filter and sterilize.
The lymphocyte culture medium utilizes a sodium glycerophosphate/HEPES buffer system, has no effect on the subsequent culture of the cells, and can stabilize the pH of the cells; the buffer system is not affected by the carbon dioxide in the environment and the remaining gas components, and is beneficial to the long-term preservation of the medium.
[1] Lymphocyte culture medium with stable pH and preparation method and application thereof. CN104988120B, 2018-12-18.
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