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Kit and method for primate neuron separation

A primate and kit technology, applied in the field of biological cells, can solve the problem of no adult primate neuron separation, etc., and achieve the effect of optimizing the separation steps

Active Publication Date: 2021-12-03
ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the special structure of brain tissue, there are no reports on the isolation of neurons in adult primates

Method used

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  • Kit and method for primate neuron separation
  • Kit and method for primate neuron separation
  • Kit and method for primate neuron separation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] A kit for isolating primate neurons according to an embodiment of the present invention includes artificial cerebrospinal fluid and brain tissue repair fluid. Wherein the preparation method of artificial cerebrospinal fluid is: mix 62.5mM / L NaCl, 12.5mM / L NaHCO 3 , 0.5mM / LNaH 2 PO 4, 1mM / L KCl, 12.5mM / L D-glucose, 1mM / LCaCl 2 , 0.5mM / L MgCl 2 , 2.5mM / L L-sodium ascorbate, 1.5mM / L sodium pyruvate, 0.01mM / L taurine, 2mM / L thiourea, 7.5mM / L trehalose, 0.5mM / L kynuric acid, 10μM / L 6 -Cyano-7-nitroquinoxaline-2,3-dione, 50 μM / L DL-AP5, 0.5 μM / L tetrodotoxin, 5 μg / ml actinomycin D were mixed evenly and continued oxygenation (95% O 2 +5%CO 2 ) for 30 minutes, and adjust the pH value of the artificial cerebrospinal fluid to 7.4 to obtain the artificial cerebrospinal fluid; the preparation method of the brain tissue repair solution is: mix 47.5mM / L N-methyl-D-glucosamine, 15mM / L NaHCO 3 , 0.6mM / L NaH 2 PO 4 , 1.25mM / L KCl, 10mM / L 4-hydroxyethylpiperazineethanesulfonic ac...

Embodiment 2

[0052] A kit for isolating primate neurons according to an embodiment of the present invention includes artificial cerebrospinal fluid and brain tissue repair fluid. Wherein the preparation method of artificial cerebrospinal fluid is: mix 60mM / L NaCl, 10mM / L NaHCO 3 , 0.4mM / LNaH 2 PO 4 , 0.9M / L KCl, 11mM / L D-glucose, 0.9mM / LCaCl 2 , 0.4mM / L MgCl 2 , 2mM / L L-sodium ascorbate, 1.25mM / L sodium pyruvate, 0.0075mM / L taurine, 1.75mM / L thiourea, 6.5mM / L trehalose, 0.4mM / L kynuric acid, 5μM / L 6 -Cyano-7-nitroquinoxaline-2,3-dione, 45 μM / L DL-AP5, 0.4 μM / L tetrodotoxin, 3.5 μg / ml actinomycin D were mixed evenly and continued oxygenation (95% o 2 +5%CO 2 ) for 30 minutes, and adjust the pH value of the artificial cerebrospinal fluid to 7.2 to obtain the artificial cerebrospinal fluid; the preparation method of the brain tissue repair solution is: mix 45mM / L N-methyl-D-glucosamine, 14mM / LNaHCO 3 , 0.5mM / L NaH 2 PO 4 , 1mM / L KCl, 9mM / L 4-hydroxyethylpiperazineethanesulfonic acid,...

Embodiment 3

[0069] A kit for isolating primate neurons according to an embodiment of the present invention includes artificial cerebrospinal fluid and brain tissue repair fluid. Wherein the preparation method of artificial cerebrospinal fluid is: mix 65mM / L NaCl, 15mM / L NaHCO 3 , 0.6mM / LNaH 2 PO 4 , 1.1mM / L KCl, 14mM / L D-glucose, 1.1mM / LCaCl 2 , 0.6mM / L MgCl 2 6 -Cyano-7-nitroquinoxaline-2,3-dione, 55 μM / L DL-AP5, 0.6 μM / L tetrodotoxin, 5.5 μg / ml actinomycin D were mixed evenly and continued oxygenation (95% o 2 +5%CO 2 ) for 30 minutes, and adjust the pH value of the artificial cerebrospinal fluid to 7.4 to obtain the artificial cerebrospinal fluid; the preparation method of the brain tissue repair solution is: mix 50mM / L N-methyl-D-glucosamine, 16mM / LNaHCO 3 , 0.7mM / L NaH 2 PO 4 , 1.5mM / L KCl, 11mM / L 4-Hydroxyethylpiperazineethanesulfonic acid, 14mM / L D-glucose, 2.75mM / L L-sodium ascorbate, 1.1mM / L Thiourea, 1.6mM / L Pyruvate Sodium, 5.75mM / L MgSO 4 , 0.3mM / L CaCl 2 After mix...

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Abstract

The invention discloses a kit and a method for primate neuron separation, and relates to the technical field of biological cells. According to the kit disclosed by the invention, the formulas of the artificial cerebrospinal fluid and the brain tissue repairing fluid are optimized, and the inhibitor mixture is added into the artificial cerebrospinal fluid, so that the survival rate of neurons is increased, the neurotoxicity is reduced, and the kit is more suitable for separating the brain cells of primates. According to the kit, the single neuron of the primate animal is successfully separated for the first time, survival of more than 90% of the neuron can be guaranteed, cell bodies, dendrites, axons, other protrusions and the like of the neuron can be reserved, almost all information of the single neuron of the primate animal can be detected, heterogeneity of different neuron can be deeply known, and more comprehensive and complete information of neurons is explored.

Description

technical field [0001] The invention relates to the technical field of biological cells, in particular to a kit and method for isolating primate neurons. Background technique [0002] In recent years, various single-cell technologies have begun to develop rapidly. After lysing and homogenizing fresh or frozen tissues and cells, single cells or single nuclei are finally obtained by separation and purification, and are applied to various single cell technologies to explore cell development, cell types and states, human diseases and stem cell technology. develop. Single cell nucleus isolation technology can obtain single nucleus from tissues that are difficult to obtain, tissue that is difficult to separate single cells, or frozen tissue, but loses cytoplasmic information such as genetic material and protein. Single-cell separation technology can obtain single cells from fresh tissues, but in some cases, cells are difficult to completely separate, such as neurons in the prima...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0793
CPCC12N5/0619C12N2509/00C12N2509/10C12N2500/05C12N2500/34C12N2500/30C12N2500/33C12N2500/38C12N2501/999C12N2501/06C12N2501/91C12N2501/734C12N2501/73C12N2501/998
Inventor 刘胜韦佳如
Owner ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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