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Method for preparing cynoglossus semilaevis testis single-cell suspension

A single-cell suspension, semi-smooth tongue sole technology, applied in the field of marine fish cell biology, can solve the problems of restricting semi-smooth tongue sole sperm, lack of experimental methods, etc., and achieve the effect of low-cost and high-efficiency acquisition

Active Publication Date: 2022-04-22
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still a lack of a good experimental method to obtain a single cell suspension of testis of semi-smooth tongue sole with suitable cell concentration and high vitality, which restricts the in-depth study of the spermatogenesis process of semi-smooth tongue sole

Method used

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  • Method for preparing cynoglossus semilaevis testis single-cell suspension
  • Method for preparing cynoglossus semilaevis testis single-cell suspension

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1 A kind of method for preparing semi-smooth tongue sole testis single-cell suspension, concrete steps are as follows:

[0025] Step 1: dissect out the testis of half-smooth tongue sole, and place it on a sterile petri dish. After peeling off other tissues adhering to the testis tissue with tweezers, wash the testis tissue with 5ml DMEM-Nacl solution 5 times, and use a blade to culture Mince the tissue on a dish to form a tissue homogenate. The preparation method of the DMEM-Nacl solution for the testis tissue washing liquid of semi-smooth tongue sole is as follows: add 1.5 g Nacl to 50 ml DMEM, shake and mix until the Nacl is completely dissolved, and use it after filtering through a filter with a pore size of 0.22 μm.

[0026] Step 2: Transfer the tissue homogenate to a 5ml centrifuge tube containing 2-4ml of enzyme mixture, mix upside down to prevent the tissue pieces from sticking to each other, and place the centrifuge tube in a 30°C water bath for enzym...

Embodiment 2

[0030] Embodiment 2 A method for preparing semi-smooth tongue sole testis single-cell suspension, the specific steps are as follows:

[0031] Step 1: dissect out the testis of half-smooth tongue sole, and place it on a sterile petri dish. After peeling off other tissues adhering to the testis tissue with tweezers, wash the testis tissue with 5ml DMEM-Nacl solution 5 times, and use a blade to culture Mince the tissue on a dish to form a tissue homogenate. The preparation method of the DMEM-Nacl solution for the testis tissue washing liquid of semi-smooth tongue sole is as follows: add 1.5 g Nacl to 50 ml DMEM, shake and mix until the Nacl is completely dissolved, and use it after filtering through a filter with a pore size of 0.22 μm.

[0032] Step 2, transfer the tissue homogenate to a 5ml centrifuge tube containing 2-4ml enzyme mixture, mix upside down to prevent the tissue pieces from sticking to each other, and place the centrifuge tube in the 37 Enzymatic digestion in ℃ w...

Embodiment 3

[0037] Embodiment 3 A method for preparing semi-smooth tongue sole testis single-cell suspension, the specific steps are as follows:

[0038] Step 1: dissect out the testis of half-smooth tongue sole, and place it on a sterile petri dish. After peeling off other tissues adhering to the testis tissue with tweezers, wash the testis tissue with 5ml DMEM-Nacl solution 5 times, and use a blade to culture Mince the tissue on a dish to form a tissue homogenate. The preparation method of the DMEM-Nacl solution for the testis tissue washing liquid of semi-smooth tongue sole is as follows: add 1.5 g Nacl to 50 ml DMEM, shake and mix until the Nacl is completely dissolved, and use it after filtering through a filter with a pore size of 0.22 μm.

[0039] Step 2: Transfer the tissue homogenate to a 5ml centrifuge tube containing 2-4ml of enzyme mixture, mix upside down to prevent the tissue pieces from sticking to each other, and place the centrifuge tube in a 30°C water bath for enzymatic...

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Abstract

The invention relates to a method for preparing cynoglossus semilaevis testis single-cell suspension, and belongs to the field of cytobiology, and the method comprises the following steps: taking cynoglossus semilaevis testis tissues, flushing the tissues with a DMEM-Nacl solution, and then chopping and homogenizing; adding an enzyme mixed solution into the tissue homogenate, carrying out enzymolysis digestion in a water bath at 30 DEG C for 5-10 minutes, and observing under a microscope until tissue blocks basically disappear; filtering the digested cell suspension with a 40 [mu] m cell sieve; centrifuging the filtrate for the first time, collecting cell precipitates, adding a PBS-Nacl solution to resuspend cells, centrifuging for the second time, and repeating twice; and adding a BSA-PBS-Nacl solution with a volume ratio of 0.1% into the washed cell precipitate, and suspending the cells again to obtain the cynoglossus semilaevis testis single-cell suspension. According to the method, the cynoglossus semilaevis testis single-cell suspension is obtained, the dissociated cell concentration is ensured to be 1000-2000 cells / mu L, and the motility rate is greater than 80%.

Description

technical field [0001] The invention belongs to the technical field of marine fish cell biology, and relates to a method for preparing single cell suspension of tongue sole testis. Background technique [0002] The half-smooth tongue sole is a large benthic warm-temperate animal distributed in the coastal waters of the Bohai Sea, Yellow Sea, East my country Sea, and South China Sea. Semi-smooth tongue sole is large in size, fast in growth, strong in adaptability, small in range of activities, high in nutritional value, and delicious in taste. It is one of the important species of mariculture in my country and has a high market value. At present, my country has realized the factory culture and artificial breeding of half smooth tongue sole. Tongue sole has obvious sexual dimorphism, and the size difference between individuals of different sexes is obvious. The body weight and body length of adult females are about 2-4 times that of adult males at the same period. Therefore,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/076C12N5/071
CPCC12N5/061C12N5/0683C12N2509/10C12N2509/00
Inventor 刘响张向辉王洪岩陈建阳黄英毅卢怡芳邵长伟
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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