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A kind of preparation method of primordial germ cell slide

A technology of primordial germ cell and slices, which is applied in the field of preparation of primordial germ cell slices, can solve the problems of difficult analysis of experimental results, high production cost, poor comparability, etc., and achieve uniform cell distribution, convenient operation, and complete tissue morphology Effect

Active Publication Date: 2022-01-07
北京科跃中楷生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing primordial germ cell climbing method is as follows: put the glass slide in a petri dish, add feeder cells, after the feeder cells grow into a single layer on the glass slide, use mitomycin or γ-ray After the treatment terminates its division, add PGCs cells, after about 24 hours of cultivation, PGCs cells are adsorbed on the feeder layer cells, and the cell climbing is completed, take out the slides, wash with PBS, and fix with 4% paraformaldehyde or other The liquid is fixed and the follow-up experiment is carried out. The whole process takes more than 2 days, which is time-consuming and laborious. The whole process is cumbersome to operate and has many defects.
With the deepening of research, cell detection indicators are becoming more and more abundant, and the demand for cell slides is gradually increasing. The traditional method needs to stain each slide one by one. There will inevitably be differences in the process of climbing slices, resulting in poor comparability between each other and difficult analysis of experimental results

Method used

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  • A kind of preparation method of primordial germ cell slide

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Embodiment 1

[0020] A preparation method of primordial germ cell slides, comprising glass slides pretreatment, PGCs cell purification, washing and fixation, and specifically includes the following steps:

[0021] 1) Pretreatment of glass slides: ultrasonically clean the glass slides with water and hydrochloric acid ethanol twice, 8 min each time, then place the glass slides in piranha solution, react at 70 °C for 20 min, and then ultrasonically each time with water and ethanol. Washed twice, then placed in 3-aminopropyltrimethoxysilane solution to react for 15h, and then ultrasonically washed with ethanol for 2 times. As for the cross-linking reaction at 110°C for 3h, the primordial germ cell crawling piece was obtained. Hydrochloric acid and ethanol washing solution It can remove mildew, has bactericidal effect, easy operation, low cost, low corrosiveness and environmental pollution, high efficiency and low loss, the piranha solution used in this step has strong oxidizing property, and can...

Embodiment 2

[0030] A method for preparing primordial germ cell slides, comprising glass slide pretreatment, PGCs cell purification, washing, and fixing, specifically comprising the following steps:

[0031] 1) Pretreatment of glass slides: ultrasonically clean the glass slides with water and hydrochloric acid ethanol for 3 times, each time for 10 minutes, then place the glass slides in piranha solution, in which concentrated H 2 SO 4 and H 2 O 2 The volume ratio is 1:2.2, the piranha solution contains 0.28% oxalic acid and 0.14% D-cellobiose, reacted at 80°C for 25min, then ultrasonically cleaned three times with water and ethanol, and then placed in 3-aminopropyl reacted in the trimethoxysilane solution for 18 hours, and then ultrasonically cleaned with ethanol for 3 times, and cross-linked at 120°C for 4 hours to obtain primordial germ cell slides;

[0032] 2) PGCs cell purification: PGCs cells were purified by Percoll density gradient centrifugation method, then washed 3 times with ...

Embodiment 3

[0036] A method for preparing primordial germ cell slides, comprising glass slide pretreatment, PGCs cell purification, washing, and fixing, specifically comprising the following steps:

[0037] 1) Pretreatment of glass slides: ultrasonically clean the glass slides with water and hydrochloric acid ethanol for 4 times, each time for 12 minutes, and then place the glass slides in piranha solution, in which concentrated H 2 SO 4 and H 2 O 2 The volume ratio of the solution is 1:2.5, the piranha solution contains 0.30% oxalic acid and 0.15% D-cellobiose, reacted at 90°C for 30 minutes, then ultrasonically cleaned 4 times with water and ethanol, and then placed in 3-aminopropyl reacted in the trimethoxysilane solution for 20 hours, then ultrasonically cleaned with ethanol for 4 times, and cross-linked at 130°C for 5 hours to obtain primordial germ cell slides;

[0038] 2) Purification of PGCs cells: PGCs cells were purified by flow sorting method, then washed 4 times with PBS, c...

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Abstract

The invention discloses a method for preparing primordial germ cell slides, which comprises glass slide pretreatment, PGCs cell purification, washing, and fixation, wherein the fixation step is: placing washed PGCs cells on amino-modified glass slides In the PFA-PBS solution containing cysteine, fix at room temperature, then remove the PFA-PBS solution, and complete the cell climbing while the cells are fixed. The beneficial effects are: the preparation method of the present invention has the advantages of simple materials, cost saving, good climbing effect, uniform distribution of cells after climbing, convenient operation, time saving and the like.

Description

technical field [0001] The invention relates to the technical field of whole in situ hybridization of cephalopod embryos, in particular to a method for preparing a primordial germ cell crawling sheet. [0002] technical background [0003] Primordial germ cells (PGCs) are progenitor cells of mammalian germ cells at all levels, which are distributed in embryos for a short time in the early stage of embryonic development. It can be isolated and then inoculated on the feeder layer for culture and transformed into embryonic germ cells (Embryonic germ cells, EG). It is a pluripotent cell with the ability to proliferate indefinitely, self-renew and differentiate into cells representing the three germ layers, and the cell can also be genetically manipulated, selected and cryopreserved without losing its pluripotency. Poultry is an oviparous animal, and it is difficult to obtain a single embryo, so the study of PGCs cells is particularly important. With the establishment of the in v...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12M3/04
CPCC12M3/04
Inventor 柳静曾小敏
Owner 北京科跃中楷生物技术有限公司
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