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Immunofluorescence staining method for buffalo spermatogonial stem cell-like cells

An immunofluorescence staining, spermatogonial stem cell technology, applied in the field of stem cells and tissues, can solve the problems of difficult to meet demand and low production performance, and achieve the effects of easy observation, increased storage time, and increased cell adhesion.

Inactive Publication Date: 2019-02-15
卢克焕 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a unique species in South China, buffalo is mainly used for traditional labor. Its production performance such as milk production and meat production is low, and it is difficult to meet the current demand for animal husbandry products.

Method used

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  • Immunofluorescence staining method for buffalo spermatogonial stem cell-like cells
  • Immunofluorescence staining method for buffalo spermatogonial stem cell-like cells
  • Immunofluorescence staining method for buffalo spermatogonial stem cell-like cells

Examples

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Embodiment 1

[0032] A method for immunofluorescence staining of buffalo spermatogonial stem cell-like cells, comprising the following steps:

[0033] (1) Suspension preparation: take buffalo testis and cut it into pieces, add 1% by mass collagenase and 0.001% by mass DNase I enzyme to digest for 5 minutes, resuspend and filter off the supernatant after digestion, and then add 0.1% Trypsin-EDTA and 0.003% DNase I were digested for 5 min, resuspended again and filtered to remove the supernatant, and the spermatogonial stem cells inside the fine tubules of the bottom resuspended solution were released. After neutralization of serum IMDM, the cells were collected into centrifuge tubes with a pipette and centrifuged, and then resuspended with culture medium and filtered with a cell mesh to obtain a single cell suspension of buffalo testis; serum was replaced by fetal bovine serum FBS and serum. Any one or a mixture of two of the KSRs, and the added amount of the serum is 10% of the IMDM.

[00...

Embodiment 2

[0040] A method for immunofluorescence staining of buffalo spermatogonial stem cell-like cells, comprising the following steps:

[0041] (1) Preparation of suspension: take buffalo testis and cut it into pieces, add 3% by mass collagenase and 0.005% by mass DNase I enzyme to digest for 15 minutes, resuspend and filter off the supernatant after digestion, and then add 0.4% Trypsin-EDTA and 0.007% DNase I enzyme were digested for 15min, resuspended again and filtered off the supernatant to obtain the bottom resuspended spermatogonial stem cells inside the fine tubules. After neutralization of serum IMDM, the cells were collected into centrifuge tubes with a pipette and centrifuged, and then resuspended with culture medium and filtered with a cell mesh to obtain a single cell suspension of buffalo testis; serum was any of the serum and serum substitute KSR. One or a mixture of two, the serum is added in an amount of 10% of IMDM.

[0042] The culture medium includes the following...

Embodiment 3

[0048] A method for immunofluorescence staining of buffalo spermatogonial stem cell-like cells, comprising the following steps:

[0049] (1) Suspension preparation: Take buffalo testis and cut it into pieces, add 2% collagenase by mass and 0.003% by mass DNase I enzyme to digest for 10 minutes, resuspend and filter off the supernatant after digestion, and then add 0.2% Trypsin-EDTA and 0.005% DNase I were digested for 10 min, resuspended and filtered to remove the supernatant, and the spermatogonial stem cells inside the fine tubules of the bottom resuspended solution were released, and blown into single cells using a pipette. After neutralization of serum IMDM, the cells were collected into centrifuge tubes with a pipette and centrifuged, and then resuspended with culture medium and filtered with a cell mesh to obtain a single cell suspension of buffalo testis; serum was replaced by fetal bovine serum FBS and serum. Any one or a mixture of two of the KSRs, and the added amoun...

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Abstract

The invention discloses an immunofluorescence staining method for buffalo spermatogonial stem cell-like cells, comprising the following steps: (1) suspension preparation: cutting buffalo testicles into pieces, performing two-step digestion, using a pipette for pipetting to obtain a single cell suspension which is then resuspended and filtered; (2) cell purification: inoculating the suspension intoa culture dish for culturing, collecting suspended and non-adherent cells, and collecting adherent cells after culturing for two times to obtain purified spermatogonial stem cell-like cells; (3) cellfixation: fixing the cells with a tissue fixing solution, spraying the fixed spermatogonial stem cell-like cells on a slide at a dose of 5[Mu]l / drop and drying; (4) incubation: drawing a circle around a specimen with a pap pen, performing permeable membrane positioning on genes in a cytoplasm or a nucleus, and incubating and washing with a primary antibody and a secondary antibody; and (5) staining: dripping a staining agent to the immunohistochemical circle, and putting a coverslip on the circle for sealing with a nail enamel seal. The immunofluorescence staining method for buffalo spermatogonial stem cell-like cells provided by the invention is easy to operate and has a good staining effect.

Description

technical field [0001] The invention relates to the technical field of stem cells and tissues, in particular to an immunofluorescence staining method for buffalo spermatogonial stem cell-like cells. Background technique [0002] Spermatogonial stem cells (SSCs) are the precursor cells that form sperm. In male mammals, the proliferation and differentiation of spermatogonial stem cells provide a steady stream of power for spermatogenesis, and at the same time ensure that the genetic material remains in the parent-child. Efficient transfer between generations ([1] Kanatsu-Shinohara et al., 2013). Since the successful development of spermatogonial stem cell transplantation in 1994 ([2] Brinster et al., 1994), the research on spermatogonial stem cells has become a hot spot, and in recent years, a method for in vitro culture of spermatogonial stem cells has been successfully established ([3] ]Shinohara et al., 2003). In 2011, Shinohara et al. successfully developed a serum-free ...

Claims

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Application Information

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IPC IPC(8): G01N1/30
CPCG01N1/30
Inventor 卢克焕李婷婷陆阳清杨小淦梁兴伟
Owner 卢克焕
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