Quick sperm acrosin extraction method and sperm acrosin activity flow cytometry detection method

An extraction method and flow detection technology, which are applied in the field of rapid extraction method and flow detection of its activity, can solve the problems of difficult accurate quantification, low acrosome enzyme activity, and insufficient sensitivity, so as to achieve improved efficiency, mild treatment, The effect of simplifying the experimental operation steps

Active Publication Date: 2017-06-27
ZHEJIANG CELLPRO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the activity of acrosome enzyme in sperm samples is not high, usually at the μIU level, there is a problem that the sensitivity is not high enough and it is difficult to accurately quantify some samples with low enzyme activity.

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  • Quick sperm acrosin extraction method and sperm acrosin activity flow cytometry detection method
  • Quick sperm acrosin extraction method and sperm acrosin activity flow cytometry detection method

Examples

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specific Embodiment 1

[0031] A method for rapidly extracting sperm acrosome enzyme, comprising the following steps:

[0032] (1) will contain 7.5*10 6 Add the semen with the number of sperm into the centrifuge tube, centrifuge at 10000rpm for 2min, absorb the seminal plasma in the tube after centrifugation, and keep the lower layer of sperm sediment;

[0033] (2) Add 200uL permeabilization solution to the centrifuge tube containing the sperm pellet, suspend the sperm pellet evenly, centrifuge at 7000rpm for 2min, remove the supernatant, then add 400uL extract solution, suspend the sperm at 37°C for 10min, and then After centrifuging at 12000rpm for 3min, collect the supernatant to obtain the acrosome enzyme extract, which contains 1.2g sodium dihydrogen phosphate, 0.2-0.4ml Triton-1000. Contains 1g of BSA, 0.35g of sodium chloride, 1.19g of sodium monohydrogen phosphate, 0.25g of sodium dihydrogen phosphate dihydrate and 0.05g of sodium azide.

[0034] The extracted acrosome can be stored at -20°...

specific Embodiment 2

[0038] A method for flow detection of sperm acrosome activity, comprising the following steps:

[0039] (1) Collection of samples: After counting the freshly collected and completely liquefied sperm samples by a sperm counter, take 7.5*10 6 Centrifuge at 10,000rpm for 2min with the number of sperm, discard the supernatant with the tip of a pipette, and leave the sperm sample to precipitate for later use;

[0040] (2) Cell lysis: Add 200uL membrane-permeating solution to the sperm cell pellet obtained in step (1), blow the cells up and down with a pipette tip to fully resuspend the sperm cells, and centrifuge the resuspension at 7000rpm for 2-4min (not If it exceeds 5 minutes, otherwise it will affect the activity of acrosome enzyme), suck off the supernatant with the tip of the pipette, and leave the precipitate for later use; the formula of the rupture solution is: every 100ml of the membrane rupture solution contains 1.2g of sodium dihydrogen phosphate, Triton-1000.2 -0.4ml...

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Abstract

The invention discloses a quick sperm acrosin extraction method and a sperm acrosin activity flow cytometry detection method. The extraction method is characterized by including: adding seminal fluid into a centrifuge tube, centrifuging to remove seminal plasma, keeping lower sperm precipitates, adding membrane rupture liquid to enable uniform suspension of sperm precipitates, centrifuging to remove supernatant, adding extracting solution to make sperms suspended, and centrifuging to collect supernatant, so that acrosin extract is obtained. The sperm acrosin activity flow cytometry detection method is characterized in that by means of polypeptide or protein microsphere coupling and polypeptide or protein fluorescence labeling, an acrosin digested substrate is subjected to fluorescence labeling and then connected to the microspheres, fluorescent microspheres in certain particle diameters can be recognized by a flow cytometry instrument, and fluorescence intensity directly reflects activity level of acrosin in a sample. By combination of quick extraction and flow cytometry, detection efficiency is greatly improved, and detection sensitivity is improved.

Description

technical field [0001] The invention relates to a technology for extracting sperm acrosome, in particular to a method for rapidly extracting sperm acrosome and a flow detection method for its activity. Background technique [0002] After human sperm enter the female reproductive tract, they must go through a period of maturation to obtain the ability to fertilize. A series of physiological changes in sperm during this period are called capacitation. After capacitation, the sperm passes through the cumulus and corona radiata, and when it reaches the zona pellucida, it undergoes sperm-egg recognition, changes in the plasma membrane, release of acrosome enzyme, enzymatic hydrolysis of the zona pellucida, and the nucleus of the sperm cell enters the egg cell and dissolves with the nucleus. The process constitutes the acrosome reaction in the broad sense, while the acrosome reaction in the narrow sense mainly includes two key processes: plasma membrane fusion induced by the zona ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/34G01N15/14
CPCG01N1/34G01N15/14
Inventor 房海燕高攀陈继勇薛志刚
Owner ZHEJIANG CELLPRO BIOTECH
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