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A kind of Nile red staining method of poultry primordial germ cells

A technology of primordial germ cells and staining method, which is applied in the field of Nile red staining of poultry primordial germ cells, can solve the problems that cannot be used as a method for cell identification, the staining effect cannot meet the requirements, and the repeatability is not ideal, etc. The effect is stable and repeatable, making up for expensive effects

Inactive Publication Date: 2018-10-12
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these three methods have various drawbacks in practice. Morphological observation is only an auxiliary observation and cannot be used as a method for cell identification. PAS can only identify PGCs that have developed to stage 4 or later (PGCs before stage 4 do not contain sugars). Original), while the antigen-antibody staining has a non-specific reaction, that is, some of the positive reactions are not PGCs, and the staining method is time-consuming and the reproducibility is not ideal.
[0005] Nile red dye is a classic dye for detecting intracellular fat droplets. It is often used for specific staining of adipocytes. When using Nile red dye for adipocyte staining, the adipocytes are generally fixed with 1.5% glutaraldehyde before staining, and then Then use 0.1-0.01mg / mL Nile Red to stain for 5 minutes; if the PGCs are directly stained by this method, the staining effect may not be satisfactory due to the differences in cell types between primordial germ cells and adipocytes Therefore, there is no relevant report on the use of Nile red dye to stain poultry primordial germ cells

Method used

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  • A kind of Nile red staining method of poultry primordial germ cells
  • A kind of Nile red staining method of poultry primordial germ cells
  • A kind of Nile red staining method of poultry primordial germ cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Obtaining Primordial Germ Cells from Poultry Blood

[0056] (1) Collection of primordial germ cells from blood

[0057] Hen eggs developed to 55-60 hours and goose eggs developed to 3.5-4 days, the blunt end was opened to expose the embryos, and the embryos were transferred to the PBS solution without calcium and magnesium ions by using a filter paper ring, gently washed, and placed on a plate placed under a microscope (40x magnification), such as figure 1 as shown, figure 1 A is the vascular distribution map of chicken embryos developed to 55-60 h, figure 1 B is the blood vessel distribution map of goose embryos developed to 3.5-4 days. The arrows in the figure show the yolk veins of chicken embryos and goose embryos; use a microneedle to draw blood from the yolk veins on both sides, and finally put it into a container filled with 10% FCS TCM199 centrifuge tubes for use.

[0058] (2) Primordial germ cells were purified by Ficoll 400 density gradient centr...

Embodiment 2

[0061] Example 2 Obtaining Primordial Germ Cells in the Gonads of Poultry

[0062] (1) Separation of gonads

[0063] Chicken embryos develop to 6-7 days, goose embryos develop to 8-9 days, the embryos are taken out from the eggs, the gonads are located above the kidneys, in strip shape, other tissues are carefully peeled off, and washed three times in PBS without calcium and magnesium ions , that is, to obtain the left and right gonads, such as image 3 shown, where image 3 A is the microscope image of chicken embryo gonad, image 3 B is the microscope image of goose embryo gonad.

[0064] (2) Digestion of gonads

[0065] Dilute 0.25% trypsin-EDTA 3 times with PBS without calcium and magnesium ions, then place the left and right gonads in the diluted trypsin-EDTA, digest at 37°C for 5 min to form single cells, and finally add DMEM to stop For digestion, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and take the precipitate;

[0066] (3) Acquisition of P...

Embodiment 3

[0069] Example 3 PGCs Nile Red staining test

[0070] (1) Dilute the 1mg / mL Nile Red / methanol solution 100 times with PBS without calcium and magnesium, add it to the PGCs cells obtained in Example 1 and Example 2, respectively, blow and beat to form a single cell in suspension, and keep away from room temperature. Light staining for 5-10 min;

[0071] (2) Centrifuge the solution at 1500rpm for 5 minutes, take the cell pellet and add PBS without calcium and magnesium to wash;

[0072] (3) Dilute the Hoechst 33342 solution with PBS without calcium and magnesium to 10 μg / ml, add it to PGCs, and stain in the dark for 2 min;

[0073] (4) Observe the cells under an inverted fluorescent microscope.

[0074] Microscope (magnification 200 times) detection results are as follows Figure 5-8 Shown are chicken blood PGCs, chicken gonad PGCs, goose blood PGCs and goose gonad PGCs, respectively. Figure 5-8 A in A is the PGCs under bright field, B is the green fluorescence of the PGCs ...

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Abstract

The invention discloses a Nile red dyeing method of poultry primordial germ cells (PGCs). The method includes: diluting Nile red / ethanol solution with PBS which does not contain calcium and magnesium ions, adding to the PGCs, blowing and beating to form suspended single cells, and dyeing for 5-10 minutes under room temperature in a light avoiding manner; centrifuging to obtain sediments, and washing with the PBS which does not contain the calcium and magnesium ions; adding diluted Hoechst 33342 solution, and dyeing for 2 minutes under room temperature in a light avoiding manner to complete the dyeing of the PGCs. The Nile red dyeing method has the advantages that Nile red is used for identifying the PGCs for the first time, the defects that existing identification methods are expensive in reagent, long in operation time, instable in experiment result, and the like are overcome, and the method is applicable to fixed PGCs and live PGCs, simple and fast in dyeing procedure, stable in dyeing effect, high in repeatability and suitable for being widely popularized and applied.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Nile red staining method for poultry primordial germ cells. Background technique [0002] Avian primordial germ cells (Primordial Germ Cells, PGCs) are a type of cells with a unique migration route. They originate from the blastoderm epiblast at the X stage, move through the hypoblast to the reproductive crescent of the primitive streak stage, and then enter the embryo The vascular system migrates with the blood circulation to settle near the reproductive primordia, and at the same time differentiates into sperm or eggs. [0003] Studies have shown that PGCs at different stages of development and cryopreserved have the ability to migrate and differentiate. Based on this, PGCs can be used to prepare germline chimeras, so that donor PGCs can differentiate into functional gametes, and then obtain homozygous PGCs Donor offspring to achieve long-term preservation and protection of poul...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/00
CPCG01N1/30
Inventor 于建宁施振旦陈哲朱欢喜闫乐艳
Owner JIANGSU ACAD OF AGRI SCI
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