Preparing method of zebrafish embryo single-cell suspension

A zebrafish embryo, single-cell suspension technology, applied in the biological field, can solve the problem that the embryo cell dispersion method cannot be used for reference, and achieve the effect of improving the quality and shortening the dispersion process.

Pending Publication Date: 2019-04-19
NANJING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It can be seen that the embryonic cell dispersion method cannot fully learn from the biological tissue (organ) dispersion method

Method used

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  • Preparing method of zebrafish embryo single-cell suspension
  • Preparing method of zebrafish embryo single-cell suspension
  • Preparing method of zebrafish embryo single-cell suspension

Examples

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Embodiment 1

[0055] A method for preparing a zebrafish embryo single-cell suspension, the following steps are performed in sequence:

[0056] (1) Take 40 zebrafish embryos and add 1 mg / mL Pronase E aqueous solution; the volume of Pronase E aqueous solution added is 3 times the volume of zebrafish embryos.

[0057] (2) Incubate at 37°C for 5 minutes.

[0058] (3) Use a 2mL disposable dropper to gently blow and aspirate the embryo to peel off the chorion, transfer it to a 1.5mL enzyme-free centrifuge tube, and quickly suck away Pronase E.

[0059] (4) Add 1.5mL E3 medium to wash, remove, and repeat 3 to 5 times (washing Pronase E).

[0060] (5) Add 500 μL of dispase pre-cooled on ice; the dispase is Dispase dissolved in HBSS at a concentration of 0.6 U / mL.

[0061] (6) Incubate at 37°C for 2 minutes, and shake intermittently during the incubation period (approximately every 15 sec).

[0062] (7) Add 100 μL FBS to stop the digestion.

[0063] (8) Pass the suspension obtained in step (7) t...

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Abstract

The invention discloses a preparing method of zebrafish embryo single-cell suspension. The preparing method sequentially comprises the following steps of 1, taking a zebrafish embryo, and adding an aqueous solution of Pronase E; 2, conducting incubation at 37 DEG C for 4-5 min; 3, utilizing a 2-mL disposable dropper for gently blow-sucking the embryo so that the chorion can be peeled off, moving the embryo into a 1.5-mL non-enzyme centrifugal tube, and quickly sucking away Pronase E; 4, adding an E3 culture medium for washing, and then removing the culture medium, wherein the step is repeated3-5 times; 5, adding dispase which is pre-cooled on ice; 6, conducting incubation at 37 DEG C for 2-5 min, wherein intermittent vibration is conducted in the incubation period; 7, adding FBS for terminating digestion; 8, making suspension obtained in step 7 pass through a 70-micrometer screen, and then making a filtrate pass through a 40-micrometer screen; 9, conducting 200-500*g centrifuging on the screened suspension for 3-5 min; 10, taking HBSS re-suspension cells which are pre-cooled on ice and contain 1% BSA; 11, conducting 200-500*g centrifuging on the suspension obtained in step 10 for3-5 min; 12, repeating steps 10 and 11 and conducting washing once; 13, taking IESC re-suspension cells pre-cooled on ice to obtain the zebrafish embryo single-cell suspension.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing zebrafish embryo single-cell suspension. Background technique [0002] With the development of sequencing technology, the cost of sequencing continues to decrease, and bio-omics technologies such as transcriptomics and genomics have been more and more applied in many fields such as medicine, environment, and food. However, traditional transcriptomics (Bulk RNA Seq) and genomics sequencing (Bulk DNA Seq) obtain the overall state of biological tissues (organs). A "homogeneous" analysis of the different types of cells that make up biological tissues (organs) is not conducive to the study of complex biological processes. Especially for immunology, oncology, and genetics research, traditional sequencing cannot obtain effective information. With the emergence of technologies such as MARS-Seq, CytoSeq, Drop-Seq, and inDrop, it is possible to sequence nuc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073
CPCC12N5/0604
Inventor 吴兵陈玲顾纬卿张徐祥任洪强
Owner NANJING UNIV
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