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Separation method of animal nervous system endothelial cell single cell

An endothelial cell and nervous system technology, which is applied in the field of separation of single cells of endothelial cells of animal nervous system, can solve problems such as preparation of a single cell suspension of non-neuronal endothelial cells, and achieves the effects of low cost, high efficiency and simple operation.

Pending Publication Date: 2022-05-17
上海纽仁生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These results suggest that single-cell sequencing technology plays an incomparable role in accurately understanding the functions of different cells in a certain brain region and the interactions between them. Methods for the preparation of single cell suspensions

Method used

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  • Separation method of animal nervous system endothelial cell single cell
  • Separation method of animal nervous system endothelial cell single cell
  • Separation method of animal nervous system endothelial cell single cell

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0061] This embodiment provides a method for isolating single cells of animal nervous system endothelial cells, comprising:

[0062] 1. Prepare a special digestion buffer for single cell dissociation: Add 44.5 mL of DMEM medium, 5 mL of FBS, 500 μL of penicillin / streptomycin double antibody and a final concentration of 2 mg / mL to a sterile 50 mL centrifuge tube. Pronase E protease and DNase I mixture with a final concentration of 25U / mL, prepared digestion buffer 6mL / tube and aliquoted, and stored at -20°C or below for future use.

[0063] 2. Prepare endothelial cell culture medium: Add 44.5 mL of DMEM medium, 5 mL of FBS, and 500 μL of penicillin / streptomycin double antibody to a sterile 50 mL centrifuge tube, and store at 4°C.

[0064] 3. After tribromoethanol anesthetized the animal, quickly cut the abdominal cavity, exposed the heart, inserted the syringe into the left ventricle, cut the right atrial appendage, perfused the heart with pre-cooled D-PBS without calcium and m...

Embodiment 2

[0075] This embodiment provides a method for isolating single cells of animal nervous system endothelial cells, comprising:

[0076] 1. Prepare a special digestion buffer for single cell dissociation: Add 44.5 mL of DMEM medium, 5 mL of FBS, 500 μL of penicillin / streptomycin double antibody and a final concentration of 1 mg / mL to a sterile 50 mL centrifuge tube. Pronase E protease and DNase I mixture with a final concentration of 30U / mL, prepared digestion buffer 6mL / tube and aliquoted, and stored at -20°C or below for future use.

[0077] 2. Prepare endothelial cell culture medium: Add 44.5 mL of DMEM medium, 5 mL of FBS, and 500 μL of penicillin / streptomycin double antibody to a sterile 50 mL centrifuge tube, and store at 4°C.

[0078] 3. After tribromoethanol anesthetized the animal, quickly cut the abdominal cavity, exposed the heart, inserted the syringe into the left ventricle, cut the right atrial appendage, perfused the heart with pre-cooled D-PBS without calcium and m...

Embodiment 3

[0089] This embodiment provides a method for isolating single cells of animal nervous system endothelial cells, comprising:

[0090] 1. Prepare a special digestion buffer for single cell dissociation: Add 44.5 mL of DMEM medium, 5 mL of FBS, 500 μL of penicillin / streptomycin double antibody and a final concentration of 3 mg / mL to a sterile 50 mL centrifuge tube. Pronase E protease and DNase I mixture with a final concentration of 20U / mL, prepared digestion buffer 6mL / tube for aliquots, and stored at -20°C or below for future use.

[0091] 2. Prepare endothelial cell culture medium: Add 44.5 mL of DMEM medium, 5 mL of FBS, and 500 μL of penicillin / streptomycin double antibody to a sterile 50 mL centrifuge tube, and store at 4°C.

[0092] 3. After tribromoethanol anesthetized the animal, quickly cut the abdominal cavity, exposed the heart, inserted the syringe into the left ventricle, cut the right atrial appendage, perfused the heart with pre-cooled D-PBS without calcium and ma...

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Abstract

The invention discloses a preparation method of an animal nervous system endothelial cell single-cell suspension, and belongs to the technical field of biology. The method comprises the following steps: carrying out mixed culture on chopped fresh nervous tissues and a cell dissociation digestion solution, centrifuging, adding an endothelial cell culture solution, and filtering to obtain a cell mixture; and mixing the cell mixture with a cell gradient separation medium, and purifying and separating the endothelial cells. The method is easy and convenient to operate, low in cost, high in efficiency and suitable for industrial production, a specific instrument is not needed, the neural endothelial cell single cell prepared through the method can be used for single cell sequencing or other analysis, and deep research on the characteristics of the neural endothelial cell is facilitated.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for separating single cells of animal nervous system endothelial cells. Background technique [0002] Blood vessels play a pivotal role in neural activity. The concept of neurovascular unit was formally proposed in a meeting of the National Institute of Neurological Disorders and Stroke in 2001. Since then, the neurovascular unit has become a hot field in neuroscience. These studies reveal that the work of neurons is a multidimensional, highly coordinated process that requires the participation of various cells and the connection of different signaling pathways. For example, the blood-brain barrier prevents neurons from being damaged by harmful substances in the blood. However, whether neuroendothelial cells, the most important vascular cells in the nervous system, interact with neurons and whether different subtypes of neuroendothelial cells participate in th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/069C12N2509/10C12N2509/00
Inventor 张允斌张倩
Owner 上海纽仁生物医药科技有限公司
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