Enzymatic hydrolysate suitable for enzymolysis of mouse brain tissue, cell separation method and application of enzymatic hydrolysate

An enzymatic hydrolysate and brain tissue technology, applied in the biological field, can solve the problems of mining data, unable to fully understand the internal mechanism of brain tissue, changing the preference of tissue dissociation, etc., and achieve the effect of high yield

Pending Publication Date: 2022-03-15
SHANGHAI OE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The key point is the formula of the enzymatic solution. Changes in the formula of the enzymatic solution will change the preference of tissue dissociation. For single-cell sequencing, the preference of dissociation will make it impossible for those skilled in the art to dig deeper into the data comprehensively. , unable to fully understand the inner mechanism of brain organization

Method used

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  • Enzymatic hydrolysate suitable for enzymolysis of mouse brain tissue, cell separation method and application of enzymatic hydrolysate
  • Enzymatic hydrolysate suitable for enzymolysis of mouse brain tissue, cell separation method and application of enzymatic hydrolysate
  • Enzymatic hydrolysate suitable for enzymolysis of mouse brain tissue, cell separation method and application of enzymatic hydrolysate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Cell isolation experiment of brain tissue of newborn 1-day mouse

[0067] 1. Enzyme solution configuration: papain 10U / ml, collagenase I 0.1% (m / v), DNase I 30U / ml. HBSS buffer (calcium ion, magnesium ion) was used as enzymolysis buffer for enzymolysis, and the prepared enzymolysis solution was filtered through a 0.22 μm filter membrane for use.

[0068] 2. Tissue cleaning: After dissecting the mouse and taking out the brain tissue, rinse the surface of the tissue 5 times with pre-cooled DPBS to remove contaminated blood, hair, grease, etc.

[0069] 3. Tissue enzymatic hydrolysis: cut the tissue into 1mm with iris scissors 3 For the left and right small pieces, add 6mL of enzymatic hydrolysis solution, place in a shaker at 37°C with a rotation speed of 90rpm, and enzymatically hydrolyze for 30min.

[0070] 4. Assisted release, add DPBS containing 20% ​​(v / v) FBS fetal bovine serum 1:1 to terminate the enzymatic hydrolysis, use a wide mouth pipette to pipette the suspe...

Embodiment 2

[0079] 12-week mouse brain tissue cell isolation experiment

[0080] 1. Enzyme solution configuration: papain 10U / ml, collagenase I 0.1% (m / v), DNase I 30U / ml. HBSS buffer (calcium ion, magnesium ion) was used as enzymolysis buffer for enzymolysis, and the prepared enzymolysis solution was filtered through a 0.22 μm filter membrane for use.

[0081] 2. Tissue cleaning: After dissecting the mouse and taking out the brain tissue, rinse the surface of the tissue 5 times with pre-cooled DPBS to remove contaminated blood, hair, grease, etc.

[0082] 3. Tissue enzymatic hydrolysis: cut the tissue into 1mm with iris scissors 3 For the left and right small pieces, add 6mL of enzymatic hydrolysis solution, place on a shaker at 37°C with a rotation speed of 90rpm, and enzymatically hydrolyze for 30min.

[0083] 4. Assisted release, add DPBS containing 20% ​​(v / v) FBS fetal bovine serum 1:1 to stop the enzymatic hydrolysis, use a wide mouth pipette to suck the suspension 10 times to he...

Embodiment 3

[0092] 1-Day Mouse Brain Tissue Cell Isolation Experiment

[0093] 1. Enzyme solution configuration: papain 20U / mL, collagenase I 0.2% (m / v), DNase I 75U / mL. HBSS buffer (calcium ion, magnesium ion) was used as enzymolysis buffer for enzymolysis, and the prepared enzymolysis solution was filtered through a 0.22 μm filter membrane for use.

[0094] 2. Tissue cleaning: After dissecting the mouse and taking out the brain tissue, rinse the surface of the tissue 5 times with pre-cooled DPBS to remove contaminated blood, hair, grease, etc.

[0095] 3. Tissue enzymatic hydrolysis: cut the tissue into 1mm with iris scissors 3 For the left and right small pieces, add 6mL of enzymatic hydrolysis solution, place on a shaker at 37°C with a rotation speed of 90rpm, and enzymatically hydrolyze for 45min.

[0096]4. Assisted release: Add DPBS containing 20% ​​(v / v) FBS fetal bovine serum 1:1 to stop the enzymatic hydrolysis, use a wide mouth pipette to suck the suspension 15 times to help ...

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Abstract

The invention discloses an enzymatic hydrolysate suitable for mouse brain tissue cell separation in any period and an optimization method for realizing low loss of a single-cell suspension. The proportion of living cells in the single-cell suspension obtained by the method is superior to that of the single-cell suspension obtained by the method in the prior art. Due to the advantages, technicians in the field can complete separation of brain tissue cells of mice in all stages and obtain cell suspension with high motility rate, high yield and low impurity ratio, or the separated cells can be continuously applied to subsequent single cell sequencing experiments, and a more real and meaningful result is obtained. And a more complete guarantee is provided for further clarification of disease mechanisms and treatment of diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for preparing an enzymolysis solution suitable for enzymolysis of mouse brain tissue, a single-cell suspension, and an application thereof. Background technique [0002] Since the advent of single-cell sequencing technology, it has had a subversive impact on life science research, revolutionizing research paradigms and basic methods in many fields, such as immunobiology, tumor biology, and developmental biology. In brain science research, single-cell sequencing has brought detailed data to analyze the functions of neurons in specific physiological and pathological states, and to identify the role of different cells in development or disease progression, revealing profound molecular mechanisms, and also It has become the most powerful technical means in brain science research. The process includes four steps: sample preparation, single cell isolation, sequencing, and data anal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64C12N9/50C12N9/22C12N5/079C12Q1/24C12Q1/6869
CPCC12N9/63C12N9/6489C12N9/22C12N5/0618C12Q1/24C12Q1/6869C12Y304/22002C12N2509/00C12N2509/10C12Q2527/125C12Q2535/122
Inventor 范黎明殷昊朱燕敏王佳琦肖云平
Owner SHANGHAI OE BIOTECH CO LTD
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