Cell sorting magnetic bead, synthesis method thereof and application thereof in cell sorting

A technology of magnetic beads and cells, applied in the field of nano-biological applications in materials science and biochemistry, can solve the problems of small protein coupling, high separation cost and complicated operation.

Inactive Publication Date: 2010-11-24
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the commonly used commercial micron-sized magnetic beads have a small amount of protein coupling, while nano-sized magnetic beads are not easy to be separated by magnets due to their small magnetism, and the operation is complicated and the separation cost is high.

Method used

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  • Cell sorting magnetic bead, synthesis method thereof and application thereof in cell sorting
  • Cell sorting magnetic bead, synthesis method thereof and application thereof in cell sorting
  • Cell sorting magnetic bead, synthesis method thereof and application thereof in cell sorting

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Fe 3 o 4 Preparation and Characterization of @Au Nanoparticles

[0036] 1. Fe 3 o 4 Preparation of (15nm) nanoparticles

[0037] a.5.2g FeCl 3 6H 2 O, 2.0g FeCl 2 4H 2 O was dissolved in 40mmol / L HCl aqueous solution to make iron salt solution;

[0038] b. Under sufficient mechanical stirring, add the prepared iron salt solution dropwise into 250mL 1.5mol / L NaOH aqueous solution, and keep the temperature at 80°C during the whole process;

[0039] c. Fe produced 3 o 4 Nanoparticles were separated by a magnet, collected and washed 4 times with 200mL pure water, dispersed in 100mL pure water to make 0.1mol / L Fe 3 o 4 storage solution.

[0040] 2. Fe 3 o 4 Preparation of @Au(50-70nm) nanoparticles ( figure 1 )

[0041] a.5mL 0.1mol / L Fe 3 o 4 Stock solution and 1.5mL 0.2mol / L NH 2 Add OH HCl aqueous solution dropwise to 75mL 0.01mol / L TMAOH aqueous solution and heat to 80°C;

[0042] b. Under full stirring, 40mL 0.1% (w / v) HAuCl 4 Add the ...

Embodiment 2

[0046] Example 2: Preparation and purification of mouse CD3 monoclonal antibody

[0047] 1. Preparation of mouse CD3 monoclonal antibody

[0048] a. Place the anti-mouse CD3 monoclonal antibody hybridoma cell line in the cell culture medium at 37°C and 5% CO 2 , change the cell culture medium every 2 days until the cell concentration is greater than 10 5 Stop changing the liquid when each hole is used;

[0049] b. Inject paraffin oil into the peritoneal cavity of healthy Balb / c mice several days before antibody mass production. Blow down a certain number of monoclonal cells with sterile PBS solution, centrifuge at 1,000rpm for 3-5min, resuspend the cell pellet in 0.5mL PBS, and inject it into the abdominal cavity of the mouse;

[0050] c. 7-10 days after the monoclonal cells were injected into the mouse, the abdomen of the mouse was obviously swollen, and the ascites was collected. Fix the mouse with the left hand, disinfect its abdomen with 75% alcohol, then place a test ...

Embodiment 3

[0058] Example 3: Directional coupling of mouse CD3 antibody on magnetic beads ( Figure 6 )

[0059] 1. Fe 3 o 4 Surface carboxylation of @Au nanoparticles

[0060] a. Weigh 25mg Fe 3 o 4 @Au nanoparticles were ultrasonically dispersed in 10mL ethanol solution containing 20mmol / L 16-mercaptohexadecanoic acid, and ultrasonically reacted for 48h;

[0061] b. After the reaction, wash the magnetic bead solution with ethanol for 3 times, centrifuge at 9,000rpm for 15min, and redisperse into 12.5mL ethanol to prepare a 2mg / mL carboxylated magnetic bead dispersion.

[0062] 2. Coupling of CD3 antibody on magnetic beads

[0063] a. Take 500μL 2mg / mL carboxylated magnetic bead dispersion, discard the ethanol after magnetic separation, redisperse in 1mL aqueous solution containing 20mg / mL EDC and 10mg / mL NHS, ultrasonically react for 1h, every 5min during the reaction Mix the reaction solution thoroughly once;

[0064] b. Separate the activated magnetic beads with a magnet and ...

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PUM

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Abstract

The invention provides a new cell sorting magnetic bead, which is a 50-70 nm gold plated Fe3O4 magnetic nanoparticle (Fe3O4 and Au nanoparticle) ostensibly coupled with proteinA by covalent bonds, interacted with the specificity of a Fc section of IgG through the proteinA, and directionally coupled with IgG type mouse CD3 antibodies. The invention relates to a method for synthesizing and modifying the magnetic bead, and a method for carrying out cell sorting by using the magnetic bead. The magnetic bead of the invention is used for removing the CD3 T cells in the mouse splenocytes so as to reduce the CD3 T cell content of the splenocytes from 36.5 percent to 0.7 percent, and has extremely high sorting efficiency. The magnetic bead also has the advantages of high protein-coupled quantity and superparamagnetism, and the preparation method has the advantages of simpleness, environmental protection, low cost and good application prospect.

Description

technical field [0001] The invention relates to a novel magnetic bead for cell sorting, including a method for synthesizing and modifying the magnetic bead, and a method for using the novel magnetic bead for cell sorting, and belongs to the technical field of nanobiological application in material science and biochemistry. Background technique [0002] Cell sorting is a commonly used technique in biology and immunology experiments. Using magnetic beads to sort cells is a common method in this field. In the method of using magnetic beads for cell sorting, the key technology is the synthesis of magnetic beads. At present, the commonly used commercial micron-sized magnetic beads have a small amount of protein coupling, while nano-sized magnetic beads are not easy to be separated by magnets due to their small magnetism, and the operation is complicated and the separation cost is high. [0003] In response to the above problems, in the field of cell sorting, a nano-scale magnetic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078
Inventor 张新祥崔毅然高晓明洪超
Owner PEKING UNIV
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