Sperm active oxygen staining kit and application method thereof
A dyeing reagent and reactive oxygen technology, applied in the field of sperm reactive oxygen staining kits, can solve the problems of inability to intuitively and accurately reflect the level of sperm ROS and white blood cell ROS interference, and achieve the effect of high accuracy, strong specificity and simple operation
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Embodiment 1
[0023] Example 1: Sperm Reactive Oxygen Staining Kit
[0024] Including dye solution A: phosphate buffer solution with pH=7.4.
[0025] Dye B: a mixed solution of DCFH-DA fluorescent probe and phosphate buffer solution with pH=7.0. The addition amount of DCFH-DA fluorescent probe in the mixed solution is 1g / 1000ml.
[0026] The preparation method of the above dye solution A is as follows: Weigh 24.7g Na respectively 2 HPO4, 3.59g NaH 2 PO4, 160.0g NaCl, placed in a 1000ml volumetric flask, add purified water to dissolve the volume to 1000ml, and obtain a phosphate buffer solution with pH=7.4.
[0027] The preparation method of dye solution B is as follows:
[0028] (1) Weigh 1.02gNa separately 2 HPO 4 , 0.39g NaH 2 PO 4 , 8.50g NaCl, placed in a 1000ml volumetric flask, add purified water to dissolve and dilute to 1000ml to obtain a phosphate buffer solution with pH=7.0;
[0029] (2) Weigh 1.0g of DCFH-DA fluorescent probe powder, place it in a 1000ml volumetric flask, add a phosphate bu...
Embodiment 2
[0030] Example 2: Use of Sperm Reactive Oxygen Staining Kit
[0031] (1) Using the dye solution A and dye solution B prepared in Example 1, 10ml dye solution A and purified water were mixed in a volume ratio of 1:19 to make 200ml working solution A; dye solution B was mixed with 2ml purified water Evenly, make working fluid B;
[0032] (2) Take 5ml working fluid A and wash 400ul semen to be tested, centrifuge at 1500rpm for 10 minutes, discard the supernatant, and make the sperm concentration 5×10 6 -10×10 6 / ml of sperm suspension;
[0033] (3) Mix 200ul sperm suspension with 100ul working solution B, and incubate at 37°C for 1.5 hours;
[0034] (4) Add 5ml of working solution A, wash, centrifuge at 1500rpm for 10 minutes, discard the supernatant, and mix well to make 2×10 6 / ml sperm suspension, take 5ul to make a smear and dry naturally;
[0035] (5) Set the excitation wavelength range to 420nm-485nm, observe the smear with a fluorescence microscope, observe 200 sperm, count the num...
Embodiment 3
[0037] Example 3: Clinical experiment report of sperm reactive oxygen species kit
[0038] Test the level of active oxygen in sperm on 500 clinical samples:
[0039] Sperm reactive oxygen staining method:
[0040] (1) Take 5ml of working solution A and wash 400ul semen to be tested, centrifuge at 1500rpm for 10 minutes, discard the supernatant, and make a sperm concentration of 5×10 6 -10×10 6 / ml of sperm suspension;
[0041] (2) Mix 200ul sperm suspension and 100ul working solution B, and incubate at 37°C for 1.5 hours;
[0042] (3) Add 5ml of working solution A, wash, centrifuge at 1500rpm for 10 minutes, discard the supernatant, mix well, and make 2×10 6 / ml sperm suspension, take 5ul to make a smear and dry naturally;
[0043] (4) Set the excitation wavelength range to 420nm-485nm, observe the smear with a fluorescence microscope, observe 200 sperm, count the number of reactive oxygen species positive sperm, the number of reactive oxygen species positive sperm greater than 20% is ab...
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