Mechanical separation method of neurons of adult mice

A technology of mechanical separation and neurons, applied in the field of biomedicine, can solve the problems of impossible mechanical separation and difficulty in the mechanism of neurotransmitter receptors in mice, and achieve the effect of simplicity, convenience, remarkable separation effect and high practicability

Inactive Publication Date: 2012-09-12
ZHEJIANG UNIV
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Problems solved by technology

[0002] The patch clamp experiment of mechanically isolated neurons has an irreplaceable role in the study of neurotransmitter receptor mechanisms. Mechanically isolated neurons have better physiological channel mechanisms than primary cultured neurons, and are usually used to Most o

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  • Mechanical separation method of neurons of adult mice
  • Mechanical separation method of neurons of adult mice
  • Mechanical separation method of neurons of adult mice

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Embodiment 1

[0021] Preparation and pretreatment of mouse hippocampal slices: 14-20 day old C57BL / 6 mice were anesthetized with pentobarbital sodium (80 mg / kg) and then decapitated. Quickly make 350-375μm thick tissue culture solution (unit mM: 125.0 NaCl, 5.0 KCl, 1.24 KH2PO4, 26.0 NaHCO3, 2.4 CaCl2, 1.3 MgSO4, 10.0 glucose) in ice bath (4°C) (control dosage 50-100ml) Tissue slices (electronic vibrating microtome VT1000S, Leica). Before mechanical separation, the slices were stored in tissue culture medium (100ml) at room temperature (24-32°C) for 30 minutes, and then in tissue preservation solution containing papain (Papain, 18U / ml, 32.2-35.3°C) for 30 minutes Afterwards (by calculation, directly add papain into the tissue preservation solution, adjust to the target concentration and target temperature (32.2-35.3°C), and then perform mechanical separation.

[0022] Mechanical separation of neurons: Place the brain slices in a 2mm cell culture dish filled with external fluid, the extern...

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Abstract

The invention which relates to the biomedicine field discloses a mechanical separation method of neurons of adult mice. The method comprises the following steps: making the adult mice's brain tissue slices with the thickness of 350-375mum in an ice bath tissue culture solution with an electronic vibration slicer, preserving for 30-60min at room temperature, putting the brain tissue slices into a papain-containing tissue culture solution under aseptic conditions, controlling the temperature at 32.2-35.3DEG C, maintaining the temperature for 30min, placing the brain slices in a cell culture dish containing an HEPES buffer solution, selecting a neural region needing separation, and separating neurons through using a cell separator; and screening healthy neurons. The method of the invention, which allows a primary culture chemical separation method to be integrated into mechanical cell separation and the brain slices to be preprocessed with the papain, makes the healthy neurons of adult and aged mice to be obtained through the mechanical separation, and has the advantages of simplicity, convenience, and substantial separation effect.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for mechanically separating adult mouse neurons. Background technique [0002] The patch clamp experiment of mechanically isolated neurons has an irreplaceable role in the study of neurotransmitter receptor mechanisms. Mechanically isolated neurons have better physiological channel mechanisms than primary cultured neurons, and are usually used to Most of the mice subjected to mechanical separation experiments were 2-3 weeks old (young). The brain structure of old mice is dense, and it is basically impossible to perform mechanical separation, which brings difficulties to the comparison of neurotransmitter receptor mechanisms in mice of different ages. Contents of the invention [0003] The purpose of the present invention is to provide a method that integrates the chemical separation method of primary culture into mechanical cell separation, treats brain slices with papain in...

Claims

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Application Information

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IPC IPC(8): C12N5/0793
Inventor 张若煜陈怀红
Owner ZHEJIANG UNIV
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