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Amphibian cell dissociation kit

An amphibian and kit technology, applied in the field of amphibian cell dissociation kits, can solve problems such as unfavorable research, uncontrollable enzyme activity, cell apoptosis and the like

Pending Publication Date: 2020-07-07
SHENZHEN HUADA GENE INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] (1) If the dissociation method of various types of mammalian cells is used, the dissociation temperature is not suitable, the activity of the enzyme cannot be controlled, and it is difficult for the cells to maintain the same state as in the body. If this is used as the research object, the accuracy of the experimental results cannot be guaranteed. authenticity;
[0006] (2) The dissociation efficiency is not high. In order to meet the research requirements, more tissues need to be dissociated, which increases the cost of the experiment;
[0007] (3) Trypsin itself has a relatively strong effect, which is easy to cause cell membrane damage and apoptosis, and it is difficult to ensure cell viability, which is not conducive to follow-up research

Method used

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Examples

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preparation example Construction

[0067] Preparation method of 1000×gentamycin solution: 350 mg of gentamicin sulfate (BBI, A620217-5G, powder) was dissolved in water and the volume was adjusted to 10 ml with water.

[0068] DPBS is Duchenne's phosphate buffer solution, which differs from commonly used standard PBS in that it does not contain calcium and magnesium ions.

Embodiment 1

[0069] Embodiment 1, the preparation of kit

[0070] The kit consists of the following components: Reagent A, Reagent B, Reagent C, Reagent D, Reagent E and Reagent F.

[0071] Reagent A is a cleaning reagent. The preparation method of reagent A: take each raw material according to Table 2, fully dissolve and mix well, then filter with a 0.22 μm filter membrane, and collect the filtrate, which is reagent A.

[0072] Table 2 (total volume 125ml)

[0073] raw material Dosage DPBS 100ml 1000×gentamicin 0.125ml 100× penicillin streptomycin 1.25ml Enzyme-free water 23.625ml

[0074] Reagent B is a pretreatment reagent. The preparation method of reagent B: EDTA-Na 2 -2H 2 Add O to reagent A, heat to dissolve, then adjust the pH value to 7.0 with sodium hydroxide, and then use reagent A to make up to 100ml, which is reagent B. In reagent B, the concentration of EDTA is 0.1g / 100ml.

[0075] Reagent C is enzyme lysate. Preparation method ...

Embodiment 2

[0079] Embodiment 2, the usage method of kit

[0080] 1. Take a sample (an organ or tissue of an amphibian, such as one of the limbs), wash it in 70% ethanol for 10 minutes, and then wash it three times in reagent A (3 minutes each time) to fully remove the attachments on the tissue surface.

[0081] 2. Take the tissue that completed step 1, place it in reagent B, soak at room temperature for 10-15 minutes, and then separate the skin tissue.

[0082] 3. Take the skin tissue obtained in step 2, cut it into pieces, put it in reagent C, soak it at room temperature for 1-3 hours, then add 2 times the volume of α-MEM medium containing 10% serum to stop the digestion, and then filter it with 70 μm cells Net filter and collect the filtrate, centrifuge the filtrate at 1000rpm for 5min, collect the cell pellet, and resuspend with α-MEM medium containing 10% serum.

[0083] 4. After completing step 2, peel off the muscle tissue from the remaining tissue, cut it into pieces, put it in r...

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Abstract

The invention discloses an amphibian cell dissociation kit which comprises the following reagents: a reagent C, a reagent D, a reagent E and a reagent F, wherein the reagent C contains collagenase I and dispase II of which the concentrations are 43000-53000U / 100ml and 90-110units / 100ml in sequence; the reagent D contains collagenase I of which the concentration is 55000-65000U / 100ml; the reagent Econtains trypsin and a metal ion chelating agent of which the concentrations are 120000-180000USPU / 100ml and 0.03-0.07g / 100ml in sequence; the reagent F contains collagenase II of which the concentration is 50000-55000U / 100ml. The invention further protects a method for dissociating amphibian tissue to obtain single cells. The method comprises the following steps: cutting skin tissue into pieces,and treating the pieces by using the reagent C; cutting muscle into pieces, firstly treating the pieces by using the reagent D, and further treating the pieces by using the reagent E; chopping bone tissue into pieces, and treating the pieces by using the reagent F. The amphibian cell dissociation kit can be also applied to research on single cells of skin, muscle and bone tissue of limbs of various amphibians.

Description

technical field [0001] The invention relates to an amphibian animal cell dissociation kit. Background technique [0002] Many amphibians represented by salamanders have strong regenerative ability, but because their own characteristics such as cell osmotic pressure and living temperature are quite different from those of mammals, their cell separation and culture methods are still waiting for breakthroughs. [0003] At present, most of the laboratories that use amphibians as experimental objects focus on the whole tissue as the research object, lack of exploration of cell heterogeneity, and stagnate on some scientific issues. And for mammals, the most common way to dissociate tissue or cultured cells into single cells is enzymatic hydrolysis, and trypsin is the most commonly used enzyme in laboratories because of its cheap price and strong effect. [0004] The problems existing in the prior art are as follows: [0005] (1) If the dissociation method of various types of mam...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0602C12N2509/00
Inventor 王晨黎瀚博刘田宾刘姗姗范广益顾颖刘心徐讯
Owner SHENZHEN HUADA GENE INST
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