Preparation method of fish single-cell suspension

A single-cell suspension, cell suspension technology, applied in the field of animal single-cell preparation, can solve the problems of low cell yield and low cell viability in single-cell suspension

Inactive Publication Date: 2017-07-07
SOUTHWEST UNIV
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  • Description
  • Claims
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Problems solved by technology

[0006] In view of this, the present application provides a method for preparing a single-cell suspension of fish in response to the above-mentioned problems. The tissue is processed more fully, which solves the problems of low cell yield and low cell viability in the preparation of fish single cell suspension

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  • Preparation method of fish single-cell suspension

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preparation example Construction

[0032] The invention provides a kind of preparation method of fish single cell suspension, comprises the following steps:

[0033] 1) Reagent preparation

[0034] 1.1) PBS reagent preparation: 0.01mol / L PBS (pH=7.2): accurately weigh 8.00gNaCl, 0.20gKCl, 1.44gNa 2 HPO 4 , 0.24gKH 2 PO 4 , dissolved in 800ml of autoclaved ultrapure water, adjusted to pH 7.2 with hydrochloric acid, adjusted to 1000ml in sterilized ultrapure water, and stored at 4°C for later use.

[0035] 1.2) D-Han's solution preparation: NaCl 8.00g, KCl 0.40g, NaCl 2 HP0 4 ·H 2 O 0.06g, KH 2 PO 4 0.06g, NaHCO 3 0.35g, 0.02g phenol red, dissolved in 800m1 high-pressure sterilized double distilled water, adjust the pH value to 7.2-7.4, sterilized ultrapure water to 1000ml, store at 4°C for later use;

[0036] 1.3) Preparation of type IV collagenase solution: Accurately weigh 50mg of type IV collagenase (Sigma company, production batch number: C012900), stir and dissolve in 100ml D-Han's solution at r...

Embodiment 1

[0042] Preparation method of single cell suspension of crucian carp kidney tissue:

[0043] 1) Reagent preparation

[0044] 1.1) PBS reagent preparation: 0.01mol / L PBS (pH=7.2): accurately weigh 8.00gNaCl, 0.20gKCl, 1.44gNa 2 HPO 4 , 0.24gKH 2 PO 4 , dissolved in 800ml of autoclaved ultrapure water, adjusted to pH 7.2 with hydrochloric acid, adjusted to 1000ml in sterilized ultrapure water, and stored at 4°C for later use.

[0045] 1.2) D-Han's solution preparation: NaCl 8.00g, KCl 0.40g, NaCl 2 HP0 4 ·H 2 O 0.06g, KH 2 PO 4 0.06g, NaHCO 3 0.35g, 0.02g phenol red, dissolved in 800m1 high-pressure sterilized double distilled water, adjust the pH value to 7.2-7.4, sterilized ultrapure water to 1000ml, store at 4°C for later use;

[0046] 1.3) Preparation of type IV collagenase solution: Accurately weigh 50mg of type IV collagenase (Sigma company, production batch number: C012900), stir and dissolve in 100ml D-Han's solution at room temperature 25°C, overnight at 4°C,...

Embodiment 2

[0062] Preparation method of single cell suspension of Taiwan loach kidney tissue:

[0063] 1) Reagent preparation

[0064] 1.1) PBS reagent preparation: 0.01mol / L PBS (pH=7.2): accurately weigh 8.00gNaCl, 0.20gKCl, 1.44gNa 2 HPO 4 , 0.24gKH 2 PO 4 , dissolved in 800ml of autoclaved ultrapure water, adjusted to pH 7.2 with hydrochloric acid, adjusted to 1000ml in sterilized ultrapure water, and stored at 4°C for later use.

[0065] 1.2) D-Han's solution preparation: NaCl 8.00g, KCl 0.40g, NaCl 2 HP0 4 ·H 2 O 0.06g, KH 2 PO 4 0.06g, NaHCO 3 0.35g, 0.02g of phenol red, dissolved in 800m1 high-pressure sterilized double distilled water, adjust the pH value to 7.2, sterilized ultrapure water to a volume of 1000ml, and store at 4°C for later use;

[0066] 1.3) Preparation of type IV collagenase solution: Accurately weigh 50mg of type IV collagenase (Sigma company, production batch number: C012900), stir and dissolve in 100ml D-Han's solution at room temperature 25°C, ov...

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Abstract

The application discloses a preparation method of a fish single-cell suspension. The preparation method comprises the following steps: preparing a PBS reagent, a D-Han liquid and an IV type collagenase liquid, preparing a kidney tissue block of a fish, and washing the kidney tissue for more than three times with PBS; putting the kidney tissue block in a flat vessel of an ice bath, adding PBS, shearing the kidney tissue block into small pieces, and putting the tissue blocks in a high throughput tissue centrifuge tube of a burnisher; adding the IV type collagenase liquid into the centrifuge tube and meanwhile, adding PBS to fix the volume to 4ml, putting a zirconium oxide wall to grind, and performing enzymatic digestion on a tissue homogenate; and performing filtration, centrifugalization, washing with PBS, performing centrifugalization to abandon supernate and removing cell debris, filtering the cell suspension and fixing the volume to 3ml with PBS, and storing the cell suspension for later use at 4 DEG C. By adopting an enzymatic hydrolysis grinding method, advantages of a griding method and an enzymatic hydrolysis method are combined. The tissue is more fully treated by enzymatic hydrolysis grinding, so that the problem that the cell yield is low and the cell viability is low for preparing the fish single-cell suspension can be solved.

Description

technical field [0001] The application belongs to the field of animal single cell preparation, in particular, it relates to a preparation method of fish single cell suspension. Background technique [0002] With the rapid development of medicine and cytoomics, the preparation of single cell suspension has become more and more important. In the research on the preparation of single cell suspension, it was the first in foreign countries to dissociate mouse teratoma tissue with trypsin to prepare single cell suspension, and it was the first in China to prepare single cell suspension of laryngeal carcinoma tissue squamous epithelial cells . Tissue single cell suspension has a wide range of uses and can be applied to cell culture, flow cytometry detection, etc. Flow cytometry (Flow Cytometry, FCM) is a technology developed in the late 1960s to quickly characterize and quantify the physical and chemical properties of cells. This technology uses a large number of single cells as ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0686C12N2509/00
Inventor 郑曙明任胜杰吴青陈智翔
Owner SOUTHWEST UNIV
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