CN110967230B - Method and kit for measuring content of active oxygen in sperm - Google Patents
Method and kit for measuring content of active oxygen in sperm Download PDFInfo
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- CN110967230B CN110967230B CN201911152181.9A CN201911152181A CN110967230B CN 110967230 B CN110967230 B CN 110967230B CN 201911152181 A CN201911152181 A CN 201911152181A CN 110967230 B CN110967230 B CN 110967230B
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 title claims abstract description 83
- 229910052760 oxygen Inorganic materials 0.000 title claims abstract description 83
- 239000001301 oxygen Substances 0.000 title claims abstract description 83
- 238000000034 method Methods 0.000 title claims abstract description 38
- 239000000243 solution Substances 0.000 claims abstract description 40
- 238000002156 mixing Methods 0.000 claims abstract description 33
- 239000012192 staining solution Substances 0.000 claims abstract description 30
- 239000007853 buffer solution Substances 0.000 claims abstract description 25
- 238000010186 staining Methods 0.000 claims abstract description 23
- 239000003085 diluting agent Substances 0.000 claims abstract description 22
- 238000006243 chemical reaction Methods 0.000 claims abstract description 21
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 claims abstract description 21
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 claims abstract description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000002245 particle Substances 0.000 claims abstract description 18
- 210000000582 semen Anatomy 0.000 claims abstract description 17
- FSVCQIDHPKZJSO-UHFFFAOYSA-L nitro blue tetrazolium dichloride Chemical compound [Cl-].[Cl-].COC1=CC(C=2C=C(OC)C(=CC=2)[N+]=2N(N=C(N=2)C=2C=CC=CC=2)C=2C=CC(=CC=2)[N+]([O-])=O)=CC=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=C([N+]([O-])=O)C=C1 FSVCQIDHPKZJSO-UHFFFAOYSA-L 0.000 claims abstract description 15
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 7
- 238000001035 drying Methods 0.000 claims abstract description 6
- 239000006228 supernatant Substances 0.000 claims abstract description 6
- 238000005406 washing Methods 0.000 claims abstract description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 39
- 238000005303 weighing Methods 0.000 claims description 23
- 238000004108 freeze drying Methods 0.000 claims description 22
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 20
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 20
- YHVQIDWAIRCSOQ-UHFFFAOYSA-N 1-nitrotetrazol-2-ium chloride Chemical compound [Cl-].[O-][N+](=O)N1C=[NH+]N=N1 YHVQIDWAIRCSOQ-UHFFFAOYSA-N 0.000 claims description 18
- 239000008176 lyophilized powder Substances 0.000 claims description 18
- 239000008055 phosphate buffer solution Substances 0.000 claims description 17
- 238000001914 filtration Methods 0.000 claims description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 13
- 239000011148 porous material Substances 0.000 claims description 12
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 10
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 10
- 239000004471 Glycine Substances 0.000 claims description 10
- 229930195725 Mannitol Natural products 0.000 claims description 10
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 10
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 10
- 239000012154 double-distilled water Substances 0.000 claims description 10
- 235000010355 mannitol Nutrition 0.000 claims description 10
- 239000000594 mannitol Substances 0.000 claims description 10
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 8
- 229940098773 bovine serum albumin Drugs 0.000 claims description 8
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 8
- 239000003223 protective agent Substances 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 7
- 238000010790 dilution Methods 0.000 claims description 6
- 239000012895 dilution Substances 0.000 claims description 6
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- ZGZLYKUHYXFIIO-UHFFFAOYSA-N 5-nitro-2h-tetrazole Chemical compound [O-][N+](=O)C=1N=NNN=1 ZGZLYKUHYXFIIO-UHFFFAOYSA-N 0.000 claims description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 3
- 239000002953 phosphate buffered saline Substances 0.000 claims 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical group P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims 1
- 210000000265 leukocyte Anatomy 0.000 abstract description 9
- 239000000843 powder Substances 0.000 abstract description 7
- 238000002360 preparation method Methods 0.000 description 10
- 238000010438 heat treatment Methods 0.000 description 8
- 238000004737 colorimetric analysis Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- 239000003642 reactive oxygen metabolite Substances 0.000 description 5
- RVBUGGBMJDPOST-UHFFFAOYSA-N 2-thiobarbituric acid Chemical compound O=C1CC(=O)NC(=S)N1 RVBUGGBMJDPOST-UHFFFAOYSA-N 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 238000007872 degassing Methods 0.000 description 4
- 238000007710 freezing Methods 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- 210000003714 granulocyte Anatomy 0.000 description 4
- 238000005086 pumping Methods 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 239000008187 granular material Substances 0.000 description 3
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 3
- 108010088751 Albumins Proteins 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 230000030120 acrosome reaction Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000004720 fertilization Effects 0.000 description 2
- 229960002897 heparin Drugs 0.000 description 2
- 229920000669 heparin Polymers 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 230000008809 cell oxidative stress Effects 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
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- 230000036541 health Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 210000004994 reproductive system Anatomy 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000008010 sperm capacitation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a method for measuring the active oxygen content of sperms, which comprises the following steps: preparing a reaction solution, mixing the diluent with nitrotetrazolium blue chloride freeze-dried powder, fully and uniformly mixing, and standing at room temperature; mixing the liquefied semen and the reaction solution, placing in a water bath box, and standing; centrifuging, removing supernatant, adding diluent, and mixing; taking the smear of the reagent obtained in the third step, dropwise adding a staining solution, reacting at room temperature, dropwise adding a staining buffer solution, uniformly mixing with the staining solution, reacting at room temperature, washing off the staining solution, and drying; and (4) observing the smear in the step four, and taking the sperms without the formazan particles as active oxygen negative sperms and the sperms with the formazan particles as active oxygen positive sperms. Observing sperms, counting the number of active oxygen positive sperms and active oxygen negative sperms respectively, and calculating the percentage of the active oxygen positive sperms; the observation result of the invention is completely not influenced by the white blood cells in the sperm, and the content of the active oxygen of the sperm to be measured can be truly reflected.
Description
Technical Field
The invention belongs to the technical field of in-vitro detection of sperm biochemical indexes, and relates to a method and a kit for measuring the content of active oxygen in sperm.
Background
The active oxygen (ROS) substance of semen is mainly produced by sperm and leucocyte in semen, and in the reproductive system, the oxidation enzyme resisting system and non-enzymatic oxidation resisting component make the ROS production and elimination keep dynamic balance. Proper amount of active oxygen plays various roles in sperm function, maintains the normal function of sperm cells, is necessary for sperm capacitation and acrosome reaction, but when ROS exists excessively, the cell oxidative stress reaction is caused, the oxidative defense system of the organism is damaged, the movement function of the sperm is changed, the vitality is reduced, the death rate is increased, and the acrosome reaction, the fertilization function and the like of the sperm are influenced.
In the prior art, methods for quantitatively measuring sperm Reactive Oxygen Species (ROS) in a laboratory include a thiobarbituric acid (TBA) method, a luminol chemiluminescence method, a horseradish peroxidase method and the like. The thiobarbituric acid (TBA) method is not strict enough in detection, and the result is not accurate, so that the method is not convenient for evaluating the condition of active oxygen; the luminol chemiluminescence method is a methodology recommended by the world health organization, but luminol with strong acid property is required to be used in the operation process, so that harm is caused to operators and the environment; the horseradish peroxidase method needs to use trichloroacetic acid which is an acidic corrosive product, also can cause harm to operators and the environment, needs ultrasonic treatment at 0 ℃, and is inconvenient for clinical use.
In order to solve the above problems, a quantitative determination kit for active oxygen species in sperm of patent application No. CN201711269951.9, the patent uses a colorimetric method to determine the content of active oxygen in sperm, and although the determination can be made, peroxidase in leukocyte can affect the determination result of active oxygen, and the patent cannot exclude the influence of leukocyte on the determination result of active oxygen content.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a method for measuring the active oxygen content of sperms.
In order to realize the purpose, the technical scheme of the invention is as follows:
a method for measuring the active oxygen content of sperm comprises the following steps:
the method comprises the following steps: preparing reaction solution, mixing 0.2-2.0ml of diluent with 20.0-300.0mg of lyophilized powder of nitrotetrazolium chloride, mixing well, and standing at room temperature for 10-30 min;
step two: mixing liquefied semen 0.1-2.0ml with the reaction solution, placing in 37 deg.C water bath box, and standing for 10-40 min;
step three: centrifuging at 200-1000g for 5-30min, discarding the supernatant, adding 0.1-2.0ml of diluent, and mixing well;
step four: taking the smear of the reagent in the third step, dripping 0.5-5.0ml of staining solution, reacting for 1-2min at room temperature, dripping 0.5-5.0ml of staining buffer solution, mixing with the staining solution, reacting for 5-30min at room temperature, washing off the staining solution, and drying;
step five: and (4) observing the smear in the step four, and taking the sperms without the formazan particles as active oxygen negative sperms and the sperms with the formazan particles as active oxygen positive sperms. Observing no less than 100 sperms, counting the number of active oxygen positive sperms and active oxygen negative sperms respectively, and calculating the percentage of the active oxygen positive sperms according to the following formula.
Further, the nitro-tetrazole blue chloride freeze-dried powder comprises the following components: comprises nitro-tetrazole blue chloride 0.02-0.3 mg/bottle, freeze-drying protective agent, excipient; wherein the freeze-drying protective agent comprises 10.0-50.0 mg/bottle of trehalose, 1.0-20.0 mg/bottle of bovine serum albumin and 0.5-3.0 mg/bottle of glycine, and the excipient comprises 5.0-100.0 mg/bottle of mannitol.
Furthermore, the capacity of the nitrotetrazolium chloride bottle is 1.0-5.0 ml.
Further, the nitro tetrazole blue chloride freeze-dried powder is prepared by the following steps: weighing 100.0mg of nitroblue tetrazolium chloride, 20.0g of trehalose, 10.0g of bovine serum albumin, 1.0g of glycine and 50g of mannitol, adding 1000ml of PBS buffer solution, stirring until the materials are completely dissolved, filtering by using filter paper with the aperture of 0.2 mu m, freeze-drying and storing at the temperature of 2-8 ℃.
Further, the diluent is phosphate buffer solution with the pH value of 6.5-8.5 and the concentration of 0.1-0.5 mmol/L.
Further, the 0.1mmol/L phosphate buffer solution is prepared by the following steps: weighing 8.5g of NaCl, 42.2HPO42 g of NaH2PO40.3g of NaH2PO40, dissolving in 900ml of double distilled water, adjusting the pH value to 7.2 by hydrochloric acid, adding water to a constant volume of 1L, filtering by using filter paper with a pore diameter of 0.1-1.0 mu m, and storing at 2-8 ℃.
Further, the staining solution is prepared by the following steps: weighing 0.1-1.0g of Rayleigh dye, adding 100.0-600.0ml of methanol, and fully and uniformly mixing until the dye is completely dissolved.
Further, the staining buffer solution is phosphate buffer solution with the pH value of 5.0-7.0 and the concentration of 0.1-0.5 mmol/L.
Further, the staining buffer is 0.1mmol/L phosphate buffer, and the preparation is carried out by the following steps: weighing NaH2PO42.12g, and Na2HPO4Dissolving 14.5g in 900ml double distilled water, adjusting pH to 6.5 with hydrochloric acid, adding water to a constant volume of 1L, filtering with filter paper with pore diameter of 0.1-1.0 μm, and storing at 2-8 deg.C.
The invention also provides a detection kit for the content of active oxygen in sperms, which has the technical scheme that:
a sperm active oxygen content detection kit comprises nitrotetrazolium chloride lyophilized powder, diluent, staining solution and staining buffer solution; the dilution is 0.1-0.5mmol/L phosphate buffer solution with pH of 6.5-8.5, the staining solution is a mixed solution of methanol and Rayleigh dye, and the staining buffer solution is 0.1-0.5mmol/L phosphate buffer solution with pH of 5.0-7.0.
Further, the kit comprises a centrifuge tube with the specification of 0.5-5.0ml, and the nitrotetrazolium chloride blue freeze-dried powder, the diluent, the staining solution and the staining buffer solution are independently filled in bottles respectively.
According to the method for measuring the active oxygen content of the sperms, the formation of the formazan particles in the sperms is observed by adopting a microscope so as to evaluate the active oxygen content of the sperms, the observation result is completely free from the influence of white blood cells in the sperm liquid, and the active oxygen content of the sperms to be measured can be truly reflected; wherein, the head acrosome area of the active oxygen negative sperm is dyed into light blue, the back area of the acrosome is dyed into blue, and the neck and the tail are dyed into light red. The active oxygen positive sperm head acrosome area is dyed into light blue, the area behind the acrosome is dyed into dark blue, the acrosome area or the area behind the acrosome can be seen to be thick purple formazan particles, the neck and the tail are dyed into light red, the sperm without the formazan particles is used as active oxygen negative sperm, and the sperm containing the formazan particles is used as active oxygen positive sperm.
Meanwhile, in the prior art, the colorimetric method for measuring the content of the active oxygen in the sperm needs colorimetric instruments such as an enzyme-labeling instrument and the like, the requirement on equipment is high, correspondingly, the cost for detecting the content of the active oxygen in the sperm is high, and the method is not beneficial to popularization in vast primary hospitals. The invention only needs a microscope, has low requirements on equipment and is suitable for popularization.
Detailed Description
In order to make those skilled in the art better understand the solution of the present invention, the following description of the technical solution in the embodiment of the present invention with reference to the examples of the present invention is clearly and completely described, and it is obvious that the described examples are only a part of examples of the present invention, and not all examples. All other embodiments obtained by a person skilled in the art based on the examples of the present invention shall fall within the scope of protection of the present invention without making creative efforts.
In the description of the present embodiments, the terms "inside", "outside", "front", "rear", "left", "right", and the like indicate orientations or positional relationships only for convenience in describing the present invention and simplifying the description, and do not indicate or imply that the referred devices or elements must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first" and "second" are used merely to distinguish similar items and are not to be construed as requiring a particular order or sequence, it being understood that such use is intended to be interchangeable where appropriate.
To clearly illustrate the design concept of the present invention, the present invention will be described with reference to the following examples.
Example 1:
a method for measuring the active oxygen content of sperm comprises the following steps:
the method comprises the following steps: preparing a reaction solution, weighing 1ml of diluent, adding the diluent into a freeze-dried powder bottle containing 100mg of nitrotetrazolium chloride, fully and uniformly mixing, and standing at room temperature for 15 min;
step two: taking 100 mu l of the reaction solution prepared in the step one, placing the reaction solution in a 2ml centrifuge tube, taking 100 mu l of liquefied semen, placing the liquefied semen in the centrifuge tube, fully and uniformly mixing the liquefied semen with the reaction solution, placing the mixture in a 37 ℃ water bath box, and placing the mixture for 30 min;
step three: centrifuging the reagent in the centrifuge tube at 250g for 5min, discarding the supernatant, adding 100 μ l of diluent, and mixing;
step four: taking 10.0 mu l smear of the reaction solution in the third step, placing the smear at room temperature until the smear is completely dried, then horizontally placing the smear, dropwise adding 2ml of staining solution on the surface of the smear, reacting at room temperature for 1min, dropwise adding 2ml of staining buffer solution to the smear, uniformly mixing with the staining solution, reacting at room temperature for 15min, washing off the staining solution with distilled water, and naturally drying;
step five: and (3) observing the smear in the step four under a microscope oil microscope, wherein the sperm without the formazan particles is taken as the active oxygen negative sperm, and the sperm containing the formazan particles is taken as the active oxygen positive sperm. Wherein, the head acrosome area of the active oxygen negative sperm is dyed into light blue, the back area of the acrosome is dyed into blue, and the neck and the tail are dyed into light red. Active oxygen positive sperm head acrosome area dyed light blue, acrosome back area dyed dark blue, acrosome or retroacrosome visible coarse purple formazan granule, neck and tail dyed light red. Observing not less than 100 sperms, respectively counting the number of active oxygen positive sperms and active oxygen negative sperms, and calculating the percentage of the active oxygen positive sperms.
In the present embodiment, the first and second electrodes are,
in the above example, the formation of the formazan particles in the sperms is observed by using a microscope to further evaluate the active oxygen content of the sperms, the observation result is completely free from the influence of white blood cells in the sperm liquid, and the active oxygen content of the sperms to be detected can be truly reflected.
Meanwhile, in the prior art, the colorimetric method for measuring the content of the active oxygen in the sperm needs colorimetric instruments such as an enzyme-labeling instrument and the like, the requirement on equipment is high, correspondingly, the cost for detecting the content of the active oxygen in the sperm is high, and the method is not beneficial to popularization in vast primary hospitals. The invention only needs a microscope, has low requirements on equipment and is suitable for popularization.
In the above example, each bottle of nitrotetrazolium chloride lyophilized powder comprises the following components: comprises nitro-blue tetrazolium chloride 0.1 mg/bottle, freeze-drying protective agent and excipient; wherein the freeze-drying protective agent comprises 20 mg/bottle of trehalose, 10 mg/bottle of albumin and 1 mg/bottle of glycine, and the excipient comprises 50 mg/bottle of mannitol, wherein the volume of the volumetric flask is 2 ml.
The nitrotetrazolium chloride lyophilized powder is prepared by the following steps: weighing 100mg of nitroblue tetrazolium chloride, 20g of trehalose, 10g of bovine serum albumin, 1g of glycine and 50g of mannitol, adding 1000ml of PBS buffer solution, stirring until the mixture is completely dissolved, filtering by using filter paper with the aperture of 0.2 mu m, and subpackaging the solution into freeze-drying bottles with the capacity of 3ml, wherein each bottle contains 1 ml. And (4) putting the subpackaged solution into a freeze dryer for freeze drying.
The freeze-drying step comprises: pre-freezing at-50 deg.C for 3h, vacuum-pumping to below 20Pa, maintaining at-50 deg.C and below 20Pa for 24 hr, heating to 0 deg.C for 4h, heating to 30 deg.C for 5h, sealing, degassing to normal pressure, and taking out to obtain nitrotetrazolium blue chloride lyophilized powder. Storing in refrigerator at 4 deg.C.
Wherein the preparation steps of the PBS buffer solution with the pH value of 7.4 are as follows: weighing NaCl 8.5g and Na2HPO42.2g, NaH2PO40.3g, dissolving in 900ml double distilled water, adjusting pH to 7.4 with hydrochloric acid, adding water to constant volume to 1L, filtering with 0.2 μm aperture filter paper, and storing at 4 deg.C for use.
The dilution is phosphate buffer solution with pH value of 6.5-8.5 and 0.1-0.5mmol/L, if the concentration of the phosphate buffer solution is lower than 0.1mmol/L or higher than 0.5mmol/L, the staining effect is poor, and the observation and determination are influenced. In this example, the diluent is a phosphate buffer solution with pH 7.2 and 0.1mmol/L, and the preparation steps are as follows: weighing NaCl 8.5g and Na2HPO42.2g,NaH2PO40.3g of the extract is dissolved in 900ml of double distilled water, the pH value is adjusted to 7.2 by hydrochloric acid, water is added to the solution until the volume is 1L, the solution can be filtered by 0.1 to 1.0 mu m pore size filter paper, in the embodiment, 0.2 mu m pore size filter paper is adopted, and the solution is stored at 4 ℃ for standby.
The dyeing liquid is a mixture of Rui's dye and methanol, and the preparation steps are as follows: weighing 0.5g of the Rui's dye, adding 300ml of methanol, and fully mixing until the dye is completely dissolved.
The staining buffer solution is phosphate buffer solution with pH value of 5.0-7.0 and 0.1-0.5mmol/L, if the concentration of the phosphate buffer solution is lower than 0.1mmol/L or higher than 0.5mmol/L, the staining effect is poor, and the observation and measurement are influenced. In this example, the staining buffer was phosphate buffer with pH of 6.5 and 0.1mmol/L, and the preparation steps were: weighing NaH2PO42.12g, and Na2HPO414.5g, dissolved in 900ml double distilled water, adjusted pH to 6.5 with hydrochloric acid, added water to a constant volume of 1L, 0.1-1.0 μm pore size filter paper can be used for filtration, in this example, 0.2 μm pore size filter paper is used, and stored at 4 ℃ for use.
In order to prove that the determination of the active oxygen can eliminate the influence of white blood cells of a sample on a detection result, the method is characterized in that the neutrophils separated from different amounts of peripheral blood are added into the same seminal plasma sample, and the active oxygen content of the sperm is determined by respectively adopting the method and a colorimetric method authorized by the patent CN201711269951.9, and the specific method comprises the following steps:
firstly, 2ml of peripheral blood of a normal person is extracted and placed in a heparin anticoagulation vacuum blood collection tube, and the mixture is inverted and mixed evenly for 10 times to ensure that the blood is fully anticoagulated.
And secondly, centrifuging the heparin anticoagulated whole blood at 2000rpm for 15min, sucking a leucocyte layer at the junction of the plasma and the erythrocyte by using a disposable pipette, placing the leucocyte layer in a 5ml glass test tube, and adding 2ml of normal saline to dilute the leucocyte layer cells.
And thirdly, taking another 5ml glass test tube, adding 2ml of the granulocyte separating medium with the specific gravity of 1.085, sucking the diluted leucocyte layer cells by a disposable pipette, adding the leucocyte layer cells on the granulocyte separating medium, and horizontally centrifuging at 2000rpm for 20 min. Sucking the granulocyte layer band on the interface of leukocyte separation liquid with disposable pipette, placing in another 5ml glass test tube, adding appropriate amount of physiological saline to adjust the concentration of the granulocyte to 10 × 106/ml。
Fourthly, taking 10ml of fresh and completely liquefied mixed semen of normal people, and adjusting the sperm density to 30 multiplied by 10 by physiological saline6/ml。
And fifthly, preparing samples with different leukocyte concentrations according to the following operation.
Sixthly, the samples No. 1 to No. 6 are respectively measured according to the counting method of the microscope and the colorimetric method authorized by the patent CN201711269951.9, and the results are as follows.
The results show that the influence of the colorimetric method for measuring the number of the leucocytes in the sample of the fertilization fluid of the active oxygen result is large, and the level of the active oxygen of the sperm cannot be objectively reflected; the microscopic counting method adopted by the technology is not influenced by the number of white blood cells in the seminal fluid specimen, and the result reliability is high.
Example two
A method for measuring the active oxygen content of sperm comprises the following steps:
the method comprises the following steps: preparing a reaction solution, weighing 2ml of a diluent, adding the diluent into a freeze-dried powder bottle containing 300mg of nitrotetrazolium chloride, fully and uniformly mixing, and standing at room temperature for 30 min;
step two: taking 100 mu l of the reaction solution prepared in the step one, placing the reaction solution in a 5ml centrifuge tube, taking 2ml of liquefied semen, placing the liquefied semen in the centrifuge tube, fully and uniformly mixing the liquefied semen with the reaction solution, placing the mixture in a 37 ℃ water bath box, and placing the mixture for 40 min;
step three: centrifuging the reagent in the centrifuge tube at 1000g for 5min, discarding the supernatant, adding 2ml of diluent, and mixing well;
step four: taking 100 mu l smear of the reaction solution in the third step, placing the smear at room temperature until the smear is completely dried, then horizontally placing the smear, dropwise adding 5ml of staining solution on the surface of the smear, reacting at room temperature for 2min, dropwise adding 2ml of staining buffer solution to the smear, uniformly mixing with the staining solution, reacting at room temperature for 30min, washing off the staining solution by using distilled water, and naturally drying;
step five: and (3) observing the smear in the step four under a microscope oil microscope, wherein the sperm without the formazan particles is taken as the active oxygen negative sperm, and the sperm containing the formazan particles is taken as the active oxygen positive sperm. Wherein, the head acrosome area of the active oxygen negative sperm is dyed into light blue, the back area of the acrosome is dyed into blue, and the neck and the tail are dyed into light red. Active oxygen positive sperm head acrosome area dyed light blue, acrosome back area dyed dark blue, acrosome or retroacrosome visible coarse purple formazan granule, neck and tail dyed light red. Observing not less than 100 sperms, respectively counting the number of active oxygen positive sperms and active oxygen negative sperms, and calculating the percentage of the active oxygen positive sperms.
This exampleIn (1),
the nitrotetrazolium chloride lyophilized powder is prepared by the following steps: weighing 300mg of nitroblue tetrazolium chloride, 50g of trehalose, 20g of bovine serum albumin, 3g of glycine and 100g of mannitol, adding 1000ml of PBS buffer solution, stirring until the solution is completely dissolved, filtering the solution by using filter paper with the aperture of 0.1 mu m, and subpackaging the solution into freeze-drying bottles with the capacity of 3ml, wherein each bottle contains 1 ml. And (4) putting the subpackaged solution into a freeze dryer for freeze drying.
The freeze-drying step is as follows: pre-freezing at-50 deg.C for 3h, vacuum-pumping to below 20Pa, maintaining at-50 deg.C and below 20Pa for 24 hr, heating to 0 deg.C for 4h, heating to 30 deg.C for 5h, sealing, degassing to normal pressure, and taking out to obtain nitrotetrazolium blue chloride lyophilized powder. Storing in refrigerator at 8 deg.C.
Wherein the staining solution is a mixture of Rui's dye and methanol, and the preparation steps are as follows: weighing 1.0g of the Rui's dye, adding 600ml of methanol, and fully mixing until the dye is completely dissolved.
The rest of the description is consistent with the embodiment.
EXAMPLE III
A method for measuring the active oxygen content of sperm comprises the following steps:
the method comprises the following steps: preparing a reaction solution, measuring 0.2ml of diluent, adding the diluent into a freeze-dried powder bottle containing 20.0mg of nitrotetrazolium chloride, fully and uniformly mixing, and standing at room temperature for 10 min;
step two: taking 100 mu l of the reaction solution prepared in the step one, placing the reaction solution in a 5ml centrifuge tube, taking 0.1ml of liquefied semen, placing the liquefied semen in the centrifuge tube, fully and uniformly mixing the liquefied semen with the reaction solution, placing the mixture in a 37 ℃ water bath box, and placing the mixture for 10 min;
step three: centrifuging the reagent in the centrifuge tube at 200g for 30min, discarding the supernatant, adding 0.1ml of diluent, and mixing well;
step four: taking 100 mu l smear of the reaction liquid in the third step, placing the smear at room temperature until the smear is completely dried, then horizontally placing the smear, dropwise adding 0.5ml of staining solution on the surface of the smear, reacting at room temperature for 1min, dropwise adding 0.5ml of staining buffer solution to the smear, uniformly mixing with the staining solution, reacting at room temperature for 5min, washing off the staining solution by using distilled water, and naturally drying;
step five: and (3) observing the smear in the step four under a microscope oil microscope, wherein the sperm without the formazan particles is taken as the active oxygen negative sperm, and the sperm containing the formazan particles is taken as the active oxygen positive sperm. Wherein, the head acrosome area of the active oxygen negative sperm is dyed into light blue, the back area of the acrosome is dyed into blue, and the neck and the tail are dyed into light red. Active oxygen positive sperm head acrosome area dyed light blue, acrosome back area dyed dark blue, acrosome or retroacrosome visible coarse purple formazan granule, neck and tail dyed light red. Observing not less than 100 sperms, respectively counting the number of active oxygen positive sperms and active oxygen negative sperms, and calculating the percentage of active oxygen positive sperms.
In the present embodiment, the first and second electrodes are,
the nitrotetrazolium chloride lyophilized powder is prepared by the following steps: weighing 20mg of nitroblue tetrazolium chloride, 10g of trehalose, 1.0g of bovine serum albumin, 0.5g of glycine and 5.0g of mannitol, adding 1000ml of PBS buffer solution, stirring until the solution is completely dissolved, filtering by using filter paper with the aperture of 1.0 mu m, and subpackaging the solution into freeze-drying bottles with the capacity of 3ml, wherein each bottle contains 1 ml. And (4) putting the subpackaged solution into a freeze dryer for freeze drying.
The freeze-drying step is as follows: pre-freezing at-50 deg.C for 3h, vacuum-pumping to below 20Pa, maintaining at-50 deg.C and below 20Pa for 24 hr, heating to 0 deg.C for 4h, heating to 30 deg.C for 5h, sealing, degassing to normal pressure, and taking out to obtain nitrotetrazolium blue chloride lyophilized powder. Storing in refrigerator at 8 deg.C.
Wherein the staining solution is a mixture of Rui's dye and methanol, and the preparation steps are as follows: weighing 0.1g of the Rui's dye, adding 100ml of methanol, and fully mixing until the dye is completely dissolved.
The rest of the description is consistent with the embodiment.
Example four
A detection kit for sperm active oxygen content comprises nitrotetrazolium chloride lyophilized powder, diluted solution, staining solution and staining buffer solution; the dilution is 0.1mmol/L phosphate buffer solution with pH of 7.2, the staining solution is a mixed solution of methanol and Rayleigh dye, and the staining buffer solution is 0.1mmol/L phosphate buffer solution with pH of 6.5. The kit also comprises a 0.5-5.0ml centrifuge tube.
In the above example, each bottle of nitrotetrazolium chloride lyophilized powder comprises the following components: comprises nitro-blue tetrazolium chloride 0.1 mg/bottle, freeze-drying protective agent and excipient; wherein the freeze-drying protective agent comprises 0.02 g/bottle of trehalose, 0.01 g/bottle of albumin and 0.001 g/bottle of glycine, and the excipient comprises 0.05 g/bottle of mannitol.
The nitrotetrazolium chloride lyophilized powder is prepared by the following steps: weighing 100mg of nitroblue tetrazolium chloride, 20g of trehalose, 10g of bovine serum albumin, 1g of glycine and 50g of mannitol, adding 1000ml of PBS buffer solution, stirring until the solution is completely dissolved, filtering the solution by using filter paper with the aperture of 0.2 mu m, and subpackaging the solution into 3ml freeze-drying bottles with 1ml of each bottle. And (4) putting the subpackaged solution into a freeze dryer for freeze drying.
The freeze-drying step is as follows: pre-freezing at-50 deg.C for 3h, vacuum-pumping to below 20Pa at-50 deg.C for 24 hr, heating to 0 deg.C for 4h, heating to 30 deg.C for 5h, sealing, degassing to normal pressure, and taking out to obtain nitrotetrazolium blue chloride lyophilized powder. Storing in refrigerator at 2-8 deg.C.
Wherein the preparation steps of the PBS buffer solution with the pH value of 7.4 are as follows: weighing 8.5g of NaCl, 42.2HPO42 g of NaH2PO40.3g of NaH2PO40, dissolving in 900ml of double distilled water, adjusting the pH value to 7.4 by using hydrochloric acid, adding water to a constant volume of 1L, filtering by using filter paper with a pore diameter of 0.2 mu m, and storing at 4 ℃ for later use.
The dilution with pH 7.2 was 0.1mmol/L phosphate buffer solution, 8.5g NaCl, 42.2HPO42 g NaH2PO40.3g was weighed out and dissolved in 900ml double distilled water, the pH was adjusted to 7.4 with hydrochloric acid, water was added to a constant volume of 1L, and the solution was filtered through 0.2 μm pore size filter paper and stored at 4 ℃ for further use.
The dyeing liquid is a mixture of Rui's dye and methanol, and the preparation steps are as follows: weighing 0.5g of the Rui's dye, adding 300ml of methanol, and fully mixing until the dye is completely dissolved.
The staining buffer solution with pH 6.5 is 0.1mmol/L phosphate buffer solution, and the preparation steps are as follows: weighing NaH2PO42.12 g and Na2HPO414.5 g, dissolving in 900ml double distilled water, adjusting pH to 6.5 with hydrochloric acid, adding water to constant volume to 1L, filtering with 0.2 μm pore size filter paper, and storing at 4 deg.C for use.
It should be noted that some of them may be selected differently than the specific examples given above. These are made by those skilled in the art based on their basic skills in understanding the concept of the present invention and are not to be considered as examples herein.
Finally, it is to be understood that the above embodiments are merely exemplary embodiments taken to illustrate the principles of the present invention, which is not intended to be limiting. It will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention, and these changes and modifications are to be considered as within the scope of the invention.
Claims (8)
1. A method for measuring the active oxygen content of sperm is characterized by comprising the following steps:
the method comprises the following steps: preparing reaction solution, mixing 0.2-2.0ml of diluent with 20.0-300.0mg of lyophilized powder of nitrotetrazolium chloride, mixing well, and standing at room temperature for 10-30 min;
step two: mixing liquefied semen 0.1-2.0ml with the reaction solution, placing in 37 deg.C water bath box, and standing for 10-40 min;
step three: centrifuging the reagent obtained in the step two for 5-30min at the concentration of 200-1000g, removing the supernatant, adding 0.1-2.0ml of diluent, and mixing uniformly;
step four: taking the reagent smear in the third step, and dripping 0.5-5.0ml of staining solution, wherein the staining solution is prepared by the following steps: weighing 0.1-1.0g of Reye dye, adding 100.0-600.0ml of methanol, and fully and uniformly mixing until the dye is completely dissolved; reacting at room temperature for 1-2min, adding 0.5-5.0ml of staining buffer solution dropwise, mixing with the staining solution, reacting at room temperature for 5-30min, washing off the staining solution, and drying; wherein the staining buffer solution is a phosphate buffer solution with the pH value of 5.0-7.0 and the concentration of 0.1-0.5 mmol/L; the 0.1mmolThe staining buffer/L was prepared by the following steps: weighing NaH2PO42.12g, and Na2HPO4Dissolving 14.5g of the extract in 900ml of double distilled water, adjusting the pH value to 6.5 by using hydrochloric acid, adding water to a constant volume of 1L, filtering by using filter paper with the pore diameter of 0.1-1.0 micron, and storing at 2-8 ℃ for later use;
step five: observing the smear in step four, taking the sperms without the formazan particles as active oxygen negative sperms, taking the sperms containing the formazan particles as active oxygen positive sperms, enabling the head vertex region of the active oxygen negative sperms to be light blue, enabling the rear region of the vertex to be dyed blue, enabling the neck and the tail to be dyed light red, enabling the head vertex region of the active oxygen positive sperms to be dyed light blue, enabling the rear region of the vertex to be dyed dark blue, enabling the large purple formazan particles to be visible in the vertex region or the rear region of the vertex, enabling the neck and the tail to be light red, observing not less than 100 sperms, respectively counting the number of the active oxygen positive sperms and the number of the active oxygen negative sperms, and calculating the percentage of the active oxygen positive sperms according to the following formula, wherein the percentage (%) of the active oxygen positive sperms is 100% of the number of the active oxygen positive sperms/the total number of the sperms.
2. The method for measuring sperm active oxygen content according to claim 1, wherein said nitrotetrazolium chloride lyophilized powder comprises the following components: comprises nitro-tetrazole blue chloride 0.02-0.3 mg/bottle, freeze-drying protective agent, excipient; wherein the freeze-drying protective agent comprises 10.0-50.0mg of trehalose per bottle, 1.0-20.0mg of bovine serum albumin per bottle and 0.5-3.0mg of glycine per bottle, and the excipient comprises 5.0-100.0mg of mannitol per bottle.
3. A method of determining the active oxygen content of sperm according to claim 1, wherein said nitrotetrazolium blue chloride is contained in a vial having a volume of 1.0-5.0 ml.
4. The method for determining the active oxygen content of sperm according to claim 2, wherein said nitrotetrazolium chloride lyophilized powder is prepared by the following steps: weighing 100.0mg of nitroblue tetrazolium chloride, 20.0g of trehalose, 10.0g of bovine serum albumin, 1.0g of glycine and 50g of mannitol, adding 1000ml of PBS buffer solution, stirring until the mixture is completely dissolved, filtering by using 0.2-micron-pore filter paper, freeze-drying, and storing at 2-8 ℃.
5. A method of determining the active oxygen content of sperm as described in claim 1, wherein said diluent is phosphate buffered saline with a pH of 6.5-8.5, 0.1-0.5 mmol/L.
6. A method as claimed in claim 5, wherein the 0.1mmol/L dilution is prepared by: weighing NaCl 8.5g and Na2HPO42.2g,NaH2PO40.3g, dissolving in 900ml double distilled water, adjusting pH to 7.2 with hydrochloric acid, adding water to constant volume to 1L, filtering with 0.1-1.0 micron pore size filter paper, and storing at 2-8 deg.C.
7. A test kit for determining the active oxygen content of sperm according to any of claims 1 to 6, wherein said kit comprises nitrotetrazolium chloride lyophilized powder, diluent, staining solution and staining buffer; the dilution is 0.1-0.5mmol/L phosphate buffer solution with pH of 6.5-8.5, the staining solution is a mixed solution of methanol and Rayleigh dye, and the staining buffer solution is 0.1-0.5mmol/L phosphate buffer solution with pH of 5.0-7.0.
8. The detection kit according to claim 7, wherein the kit comprises a centrifuge tube with the specification of 0.5-5.0ml, and the nitrotetrazolium chloride lyophilized powder, the diluent, the staining solution and the staining buffer solution are respectively and independently filled in a bottle.
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