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Method for transfecting Jurkat cells

A cell and well plate technology, applied in the biological field, can solve the problems of inapplicable Jurkat cells, waste of reagents, high concentration, etc.

Inactive Publication Date: 2017-08-08
SHANXI UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, this method does not work for Jurkat cells
The reason is that poly-L-lysine can be digested and absorbed by cells, excessive intake of poly-L-lysine will produce certain cytotoxicity, and the concentration used is very large (1mg / mL), resulting in waste of reagents

Method used

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  • Method for transfecting Jurkat cells
  • Method for transfecting Jurkat cells
  • Method for transfecting Jurkat cells

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Embodiment 1

[0017] Embodiment 1 A method of transfecting Jurkat cells, comprising the following steps:

[0018] a. Add 150 μL of PBS containing 0.1 mg / mL poly-D-lysine to the 24-well plate, add 150 μL of PBS to the other well of the 24-well plate as a control group, and let stand overnight in a 4-degree refrigerator.

[0019] b. Suck off the supernatant, wash 3 times with PBS, mix 1.5×10 6 Jurkat cells were resuspended in 500 μL of RPMI1640 medium containing 10% newborn bovine serum and antibiotic-free, and cultured in a 37-degree, 5% CO2 incubator for 12 hours. Then the unattached cells were sucked off, washed 3 times with PBS for 5 minutes each time, and 300 μL of PBS was added to observe the adhesion of Jurkat cells under a microscope ( figure 1 ). Randomly select 8 fields of view, record the number of cells in each field of view, divide the number of cells in each group by the number of cells in the control group, and obtain the relative adhesion rate.

[0020] c. When the relative...

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Abstract

The invention discloses a method for transfecting Jurkat cells. The method comprises the following steps: adding 0.1mg / mL poly-D-lysine into a 24-pore cell culture plate, standing at the temperature of 4 DEG C overnight, sucking out liquid, uncapping and blowing in a super clean bench, and washing with a PBS (Phosphate Buffered Solution); inoculating Jurkat cells into the culture plate, culturing in an incubator, removing supernatant the next day, and washing with the PBS for 3 times to turn the Jurkat cell into a wall-attaching state; transfecting pLL3.7 plasmids according to conventional transfecting steps of wall-attaching cells; observing the expression amount of GFPs (Green Fluorescent Proteins) under a fluorescence microscope 24 hours after transfection in order to determine the transfecting efficiency. By adopting the method, the contact opportunity between a plasmid compound and cells can be increased after the Jurkat cells are subjected to wall-attaching treatment, and the transfecting efficiency is increased effectively (90 percent or more). The cell viability is not influenced by a wall-attaching treatment process, and exogenous plasmids can be transfected for the Jurkat cells safely.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for transfecting Jurkat cells. Background technique [0002] Exogenous gene transfer is a technology that uses physical, chemical or biological methods to transfer exogenous genes into recipient cells and enable them to achieve gene amplification and expression in cells. It has been widely used in modern life science research and clinical gene therapy. Common exogenous gene transfer strategies mainly use calcium phosphate precipitation method, DEAE-glucose method and lipofection method, etc., especially lipofection method has been widely promoted. However, the liposome method is only suitable for adherent cells, and it is difficult to obtain satisfactory transfection efficiency for suspension cells. At present, the transfection of suspension cells mainly adopts electroporation technology, which not only requires a specific electrotransfer instrument, but also causes great ...

Claims

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Application Information

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IPC IPC(8): C12N15/85
CPCC12N15/85
Inventor 落继先王俊婷井维鑫王兰
Owner SHANXI UNIV
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