CN103822816A - Method for identifying mixed sperms by using fluorescent dyes FITC (fluorescein isothiocyanate) and MITO (Mitotracker) - Google Patents
Method for identifying mixed sperms by using fluorescent dyes FITC (fluorescein isothiocyanate) and MITO (Mitotracker) Download PDFInfo
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Abstract
The invention discloses a method for identifying mixed sperms by using fluorescent dyes FITC (fluorescein isothiocyanate) and MITO (Mitotracker), and belongs to a sperm identification method. The method comprises the steps: adding DMSO (dimethylsulfoxide) into FITC powder to prepare a FITC concentrated liquid, adding DMSO into MITO powder to prepare an MITO concentrated liquid, preparing a stainingtalp sperm dyeing buffer solution from HEPES (hydroxyethyl piperazine ethane sulfonic acid), magnesium chloride, sodium chloride, potassium chloride, disodium hydrogen phosphate, sodium hydrogen carbonate, sodium pyruvate, glucose, sodium lactate, albumin bovine serum and ultra-pure water, performing FITC dyeing and MITO dyeing on sperms, equivalently mixing the sperms subjected to FITC dyeing with the sperms subjected to MITO dyeing, and performing 3-hour fluorescent detection after FITC and MITO dyeing and the equivalent mixing of the sperms subjected to the FITC dyeing and the MITO dyeing. The method has the benefits that under the condition that the activity of the sperms is not affected, the sperms with similar forms can be identified within a certain time only through the two fluorescent dyes.
Description
Technical field
The invention belongs to a kind of sperm authentication method, particularly a kind of fluorescent dye FITC that uses identifies the method for mixing sperm with MITO.
Background technology
The sperm of different plant species has certain difference from form, can distinguish by form, but the length of close, big or small, the smart contouring head of the sperm morphology of some species, afterbody only distinguishes that by microscope tool acquires a certain degree of difficulty, after particularly two kinds of sperms being mixed, be difficult to distinguish each other again.Be exactly to distinguish by different colors for the close sperm the best way of form at present.By mixing after different dyes dyeing for different sperms, can distinguish different sperms by color separately, but fluorescent dye kind is a lot, because dyestuff does not have specificity, therefore after mixed different staining of sperm, colour contamination phenomenon very easily occurs, the sperm of dying different colours after colour contamination just becomes same color and cannot distinguish; What have has impact to sperm motility; Some phosphor persistence time is short.For this reason, need to find out one can not affect sperm motility, again not colour contamination sperm close form, that cannot distinguish by microscope is distinguished to method for distinguishing.
Summary of the invention
The object of the present invention is to provide a kind of fluorescent dye FITC that uses to identify the method for mixing sperm with MITO, this know-why is to utilize FITC to be combined and to send yellow-green fluorescence with Researches on Sperm Membrane Proteins, MITO is combined and is sent red fluorescence with mitochondria of sperms, FITC and MITO, are blended under fluorescence and can distinguish two kinds of sperms after two kinds of different sperms dye separately with two kinds of dyestuffs all without impact sperm motility.
The object of the invention is to be achieved through the following technical solutions: a kind of fluorescent dye FITC that uses identifies the method for mixing sperm with MITO, it is characterized in that: it comprises the following steps:
One, reagent preparation:
(1) the dense liquid storage of configuration FITC:
To in FITC powder, add DMSO, making its final concentration is 0.5M, and after packing, lucifuge is-20 ℃ of preservations, and term of life is 1 year;
(2) the dense liquid storage of configuration MITO:
To in MITO powder, add DMSO, making its final concentration is 0.069mM/L, and after packing, lucifuge is-20 ℃ of preservations, and term of life is 1 year;
(3) configuration staining talp staining of sperm damping fluid:
Two, staining of sperm:
(1) sperm FITC dyeing:
1. get the staining talp staining of sperm damping fluid that 5,000,000 sperms add 5ml, making sperm final concentration is 1,000,000/ml;
2. the dense liquid storage of FITC that adds 0.75 μ l in step sperm solution 1., making its final concentration is 0.077mM/L, shakes dyeing liquor is shaken up gently;
3. 40 points of 37 ℃ of lucifuge dyeing, the sperm of dyeing is jiggled;
4. after dyeing finishes, centrifugal 10 points of 1500rpm, abandons supernatant, then adds 5ml staining talp staining of sperm damping fluid, gently shake;
5. centrifugal 10 points of 1500rpm, abandons supernatant, then adds 5ml staining talp staining of sperm damping fluid, gently shake;
6. centrifugal 10 points of 1500rpm, abandons supernatant, adds 250 μ l staining talp staining of sperm damping fluids;
(2) sperm MITO dyeing:
1. get the staining talp staining of sperm damping fluid that 5,000,000 sperms add 5ml, making sperm final concentration is 1,000,000/ml;
2. the dense liquid storage of MITO that adds 1.81 μ l in step sperm solution 1., making its final concentration is 3.45 × 10
-5mM/L, shakes liquid is shaken up gently;
3. 10 points of 37 ℃ of lucifuge dyeing;
4. after dyeing finishes, centrifugal 10 points of 1500rpm, abandons supernatant, then adds 5ml staining talp staining of sperm damping fluid, gently shake;
5. centrifugal 10 points of 1500rpm, abandons supernatant, then adds 5ml staining talp staining of sperm damping fluid, gently shake;
6. centrifugal 10 points of 1500rpm, abandons supernatant, adds 250 μ l staining talp staining of sperm damping fluids;
(3) sperm mixes:
By the sperm mixed in equal amounts after the sperm after FITC dyeing and step (2) MITO dyeing for step (1), be placed on 37 ℃, 3h;
(4) detect:
1. the sperm after the sperm after FITC dyeing, step (2) MITO dyeing for step (1) is carried out to fluoroscopic examination;
2. after the sperm mixed in equal amounts after the sperm after step (3) FITC dyeing being dyeed with MITO, the sperm that mixes of 3h carries out fluoroscopic examination.
The invention has the beneficial effects as follows: do not affecting under the condition of sperm motility, only dying light by two kinds of fluorescence and just sperm close form can be distinguished within a certain period of time.
1, use these two fluorescent dyes not affect the vigor of sperm
| Sperm motility before dyeing | Sperm motility after dyeing | |
| The group of being unstained | 0.65 | |
| FITC group | 0.65 | 0.62 |
| MITO group | 0.65 | 0.6 |
Dyeing group is not remarkable with the vigor difference of the group of being unstained.
2, use this two fluorescent dyes, can the short time in colour contamination;
Two kinds of color sperms mix with 1:1, statistics after mixing 3h
| Statistics 1 | Statistics 2 | Statistics 3 | Amount to |
| Green sperm | 52 | 60 | 47 | 159 |
| Red sperm | 55 | 52 | 43 | 150 |
The also convergence 1:1 of ratio of 3h sperm after mixing.
3, colour-fast in the dyestuff short time
3h sperm fluorescence intensity change
| 1h | 2h | 3h | |
| FITC | +++ | +++ | +++ |
| MITO | +++ | +++ | +++ |
(note: "+" represents fluorescence intensity)
Accompanying drawing explanation
Fig. 1 is the rear ox sperm gray-scale map of FITC dyeing of embodiment 1;
Fig. 2 is the rear sheep sperm gray-scale map of MITO dyeing of embodiment 1;
Fig. 3 is placed on 37 ℃ after being the sheep sperm mixed in equal amounts after Niu Jingzi and the MITO dyeing after the FITC of embodiment 1 dyes, and 3h, observes gray-scale map under fluorescence;
Fig. 4 is the rear ox sperm gray-scale map of FITC dyeing of embodiment 2;
Fig. 5 is the rear ox sperm gray-scale map of MITO dyeing of embodiment 2;
Fig. 6 is placed on 37 ℃ after being the Niu Jingzi mixed in equal amounts after Niu Jingzi and the MITO dyeing after the FITC of embodiment 2 dyes, and 3h, observes gray-scale map under fluorescence.
Embodiment
Below in conjunction with embodiment, the present invention is described; the scheme of embodiment described here; do not limit the present invention; one of skill in the art can make improvements and change according to spirit of the present invention; these described improvement and variation all should be considered as in protection scope of the present invention, and scope of the present invention and essence are limited by claim.
In embodiment, medicine FITC powder used, DMSO are all purchased from SIGMA company, and MITO powder is purchased from INVITROGEN company, and the reagent not particularly pointing out is commercially available prod.
Being explained as follows of the english abbreviation letter occurring in the present invention:
1, the English name of FITC powder is: Fluorescein isothiocyanate isomer I, and sigma, F7250, Chinese is: fluorescein isothiocynate isomeride I,
2, the English name of DMSO is: DIMETHYL SULPHOXIDE, and Chinese is: dimethyl sulfoxide (DMSO),
3, the English name of MITO powder is:
red FM-Special Packaging, invitrogen, M22425, Chinese is: mitochondria red fluorescence probe,
4, the Chinese of HEPES is: 4-hydroxyethyl piperazine ethanesulfonic acid; N-(2-hydroxyethyl) piperazine-N'-2-ethane sulfonic acid.
Embodiment 1:
Use fluorescent dye FITC to identify a method of mixing sperm with MITO, it comprises the following steps:
1, reagent preparation:
(1) the dense liquid storage of configuration FITC:
To in FITC powder, add DMSO, making its final concentration is 0.5M, and after packing, lucifuge is-20 ℃ of preservations, and term of life is 1 year;
(2) the dense liquid storage of configuration MITO:
To in MITO powder, add DMSO, making its final concentration is 0.069mM/L, and after packing, lucifuge is-20 ℃ of preservations, and term of life is 1 year;
(3) configuration staining talp staining of sperm damping fluid:
2, staining of sperm:
(1) Niu Jingzi FITC dyeing:
1. get the staining talp staining of sperm damping fluid that 5,000,000 Niu Jingzi add 5ml, making sperm final concentration is 1,000,000/ml;
2. the dense liquid storage of FITC that adds 0.75 μ l in step sperm solution 1., making its final concentration is 0.077mM/L, shakes dyeing liquor is shaken up gently;
3. 40 points of 37 ℃ of lucifuge dyeing, jiggle every 10 points of sperms by dyeing;
4. after dyeing finishes, centrifugal 10 points of 1500rpm, abandons supernatant, then adds 5ml staining talp staining of sperm damping fluid, gently shake;
5. centrifugal 10 points of 1500rpm, abandons supernatant, then adds 5ml staining talp staining of sperm damping fluid, gently shake;
6. centrifugal 10 points of 1500rpm, abandons supernatant, adds 250 μ l staining talp staining of sperm damping fluids;
(2) sheep sperm MITO dyeing:
1. get the staining talp staining of sperm damping fluid that 5,000,000 sheep sperms add 5ml, making sperm final concentration is 1,000,000/ml;
2. the dense liquid storage of MITO that adds 1.81 μ l in step sperm solution 1., making its final concentration is 3.45 × 10
-5mM/L, shakes liquid is shaken up gently;
3. 10 points of 37 ℃ of lucifuge dyeing;
4. after dyeing finishes, centrifugal 10 points of 1500rpm, abandons supernatant, then adds 5ml staining talp staining of sperm damping fluid, gently shake;
5. centrifugal 10 points of 1500rpm, abandons supernatant, then adds 5ml staining talp staining of sperm damping fluid, gently shake;
6. centrifugal 10 points of 1500rpm, abandons supernatant, adds 250 μ l staining talp staining of sperm damping fluids;
(3), sperm mixes:
By the sheep sperm mixed in equal amounts after the Niu Jingzi after FITC dyeing and step (2) MITO dyeing for step (1), be placed on 37 ℃, 3h;
(4), detect:
1. step (1) is carried out to fluoroscopic examination with Niu Jingzi and sheep sperm after FITC and step (2) MITO dyeing; See that Fig. 1 is ox sperm after FITC dyeing, see that Fig. 2 is sheep sperm after MITO dyeing;
2. to after the sheep sperm mixed in equal amounts after the Niu Jingzi after step (3) FITC dyeing and MITO dyeing, be placed on 37 ℃, 3h observes under fluorescence; See Fig. 3, that green is Niu Jingzi, and red is sheep sperm.
Embodiment 2: a kind of fluorescent dye FITC that uses identifies the method for mixing sperm with MITO, and it comprises the following steps:
1, reagent preparation:
(1) the dense liquid storage of configuration FITC:
To in FITC powder, add DMSO, making its final concentration is 0.5M, and after packing, lucifuge is-20 ℃ of preservations, and term of life is 1 year;
(2) the dense liquid storage of configuration MITO:
To in MITO powder, add DMSO, making its final concentration is 0.069mM/L, and after packing, lucifuge is-20 ℃ of preservations, and term of life is 1 year;
(3) configuration staining talp staining of sperm damping fluid:
2, staining of sperm:
(1) Niu Jingzi FITC dyeing:
1. get the staining talp staining of sperm damping fluid that 5,000,000 Niu Jingzi add 5ml, making sperm final concentration is 1,000,000/ml;
2. the dense liquid storage of FITC that adds 0.75 μ l in step Niu Jingzi solution 1., making its final concentration is 0.077mM, shakes dyeing liquor is shaken up gently;
3. 40 points of 37 ℃ of lucifuge dyeing, jiggle every 10 points of Niu Jingzi by dyeing;
4. after dyeing finishes, centrifugal 10 points of 1500rpm, abandons supernatant, then adds 5ml staining talp staining of sperm damping fluid, gently shake;
5. centrifugal 10 points of 1500rpm, abandons supernatant, then adds 5ml staining talp staining of sperm damping fluid, gently shake;
6. centrifugal 10 points of 1500rpm, abandons supernatant, adds 250 μ l staining talp staining of sperm damping fluids;
(2) Niu Jingzi MITO dyeing
1. get the staining talp staining of sperm damping fluid that 5,000,000 Niu Jingzi add 5ml, making sperm final concentration is 1,000,000/ml;
2. the dense liquid storage of MITO that adds 1.81 μ l in step Niu Jingzi solution 1., making its final concentration is 3.45 × 10
-5mM, shakes liquid is shaken up gently;
3. 10 points of 37 ℃ of lucifuge dyeing;
4. after dyeing finishes, centrifugal 10 points of 1500rpm, abandons supernatant, then adds 5ml staining talp staining of sperm damping fluid, gently shake;
5. centrifugal 10 points of 1500rpm, abandons supernatant, then adds 5ml staining talp staining of sperm damping fluid, gently shake;
6. centrifugal 10 points of 1500rpm, abandons supernatant, adds 250 μ l staining talp staining of sperm damping fluids;
3, sperm mixes
By the Niu Jingzi mixed in equal amounts after the Niu Jingzi after FITC dyeing and step (2) MITO dyeing for step (1), be placed on 37 ℃, 3h;
4, detect
1. fluoroscopic examination after FITC, MITO dyeing; Fig. 4 is that the rear ox sperm of FITC dyeing and Fig. 5 are ox sperm after MITO dyeing;
2. after the Niu Jingzi mixed in equal amounts after Niu Jingzi and the MITO dyeing after FITC dyeing, be placed on 37 ℃, 3h observes under fluorescence; See Fig. 6;
Conclusion: still can distinguish after the sperm mixing 3h of two kinds of colors, all not conspiring to create identical color cannot differentiate.Therefore, in the time distinguishing the sperm of any identical or different animal, use this two kinds of fluorescent dyes, both harmless to sperm, can distinguish again different and identical sperm.
Claims (1)
1. use fluorescent dye FITC to identify a method of mixing sperm with MITO, it is characterized in that: it comprises the following steps:
One, reagent preparation:
(1) the dense liquid storage of configuration FITC:
To in FITC powder, add DMSO, making its final concentration is 0.5M, and after packing, lucifuge is-20 ℃ of preservations, and term of life is 1 year;
(2) the dense liquid storage of configuration MITO:
To in MITO powder, add DMSO, making its final concentration is 0.069mM/L, and after packing, lucifuge is-20 ℃ of preservations, and term of life is 1 year;
(3) configuration staining talp staining of sperm damping fluid:
HEPES 40mM/L
Magnesium chloride 0.4mM/L
Sodium chloride 95mM/L
Potassium chloride 3mM/L
Sodium hydrogen phosphate 0.3mM/L
Sodium bicarbonate 10mM/L
Sodium Pyruvate 2mM/L
Glucose 5mM/L
Sodium lactate 25mM/L
Bovine serum albumin(BSA) 3g/L
All the other are ultrapure water for ultrapure water;
Two, staining of sperm:
(1) sperm FITC dyeing:
1. get the staining talp staining of sperm damping fluid that 5,000,000 sperms add 5ml, making sperm final concentration is 1,000,000/ml;
2. the dense liquid storage of FITC that adds 0.75 μ l in step sperm solution 1., making its final concentration is 0.077mM/L, shakes dyeing liquor is shaken up gently;
3. 40 points of 37 ℃ of lucifuge dyeing, the sperm of dyeing is jiggled;
4. after dyeing finishes, centrifugal 10 points of 1500rpm, abandons supernatant, then adds 5ml staining talp staining of sperm damping fluid, gently shake;
5. centrifugal 10 points of 1500rpm, abandons supernatant, then adds 5ml staining talp staining of sperm damping fluid, gently shake;
6. centrifugal 10 points of 1500rpm, abandons supernatant, adds 250 μ l staining talp staining of sperm damping fluids;
(2) sperm MITO dyeing:
1. get the staining talp staining of sperm damping fluid that 5,000,000 sperms add 5ml, making sperm final concentration is 1,000,000/ml;
2. the dense liquid storage of MITO that adds 1.81 μ l in step sperm solution 1., making its final concentration is 3.45 × 10
-5mM/L, shakes liquid is shaken up gently;
3. 10 points of 37 ℃ of lucifuge dyeing;
4. after dyeing finishes, centrifugal 10 points of 1500rpm, abandons supernatant, then adds 5ml staining talp staining of sperm damping fluid, gently shake;
5. centrifugal 10 points of 1500rpm, abandons supernatant, then adds 5ml staining talp staining of sperm damping fluid, gently shake;
6. centrifugal 10 points of 1500rpm, abandons supernatant, adds 250 μ l staining talp staining of sperm damping fluids;
(3) sperm mixes:
By the sperm mixed in equal amounts after the sperm after FITC dyeing and step (2) MITO dyeing for step (1), be placed on 37 ℃, 3h;
(4) detect:
1. the sperm after the sperm after FITC dyeing, step (2) MITO dyeing for step (1) is carried out to fluoroscopic examination;
2. after the sperm mixed in equal amounts after the sperm after step (3) FITC dyeing being dyeed with MITO, the sperm that mixes of 3h carries out fluoroscopic examination.
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