CN110146431A - A kind of preparation method and cell lysis buffer solution of the flow cytometry sample suitable for Calanthe plant - Google Patents
A kind of preparation method and cell lysis buffer solution of the flow cytometry sample suitable for Calanthe plant Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 23
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- 238000004043 dyeing Methods 0.000 claims abstract description 28
- 238000005336 cracking Methods 0.000 claims abstract description 8
- 238000012545 processing Methods 0.000 claims abstract description 6
- 238000003776 cleavage reaction Methods 0.000 claims abstract description 3
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- 238000000034 method Methods 0.000 claims description 18
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- 238000005464 sample preparation method Methods 0.000 claims description 10
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- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 5
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- 239000012723 sample buffer Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
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- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 description 1
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Classifications
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- 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
-
- 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
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses the preparation methods and cell lysis buffer solution of a kind of flow cytometry sample suitable for Calanthe plant, are related to Flow cytometry field.The preparation method of flow cytometry sample disclosed by the invention suitable for Calanthe plant, tissue sample is subjected to cracking processing, filtration treatment, centrifugal treating including cleavage step, it obtains nuclear targeting step to be dyed and dyeing processing is carried out to the nucleus to be dyed, the nucleus dyed.The preparation method handles tissue sample using improved cell lysis buffer solution, it can remove in Calanthe plant remaining cytosolic fraction in complete nucleus, it keeps stabilization of the nucleus in suspension and prevents to be aggregated, and suitable environment is provided and is used to carry out single-minded nuclear chemistry dyeing, it is effectively reduced negative effect of the cytoplasmic components to dyeing.
Description
Technical field
The present invention relates to Flow cytometry fields, in particular to a kind of stream suitable for Calanthe plant
The preparation method and cell lysis buffer solution of formula cell art sample.
Background technique
Calanthe is subordinate to orchid, and leaf is big and pattern is bright-coloured, and entire inflorescence superposition is orderly, ornamental valence with higher
Value, can make potting or ground is applied to garden and Public Landscape.There are about more than 500 kinds in the Calanthe plant whole world, wherein there is 54 kinds
Native to China, long-term artificial hybridization and breeding are passed through by foreign countries, so far Calanthe kind existing more than 400.Determine some tools
There is the Genome Size of the Calanthe plant of important research value and economic characters, no matter to the Calanthe full genome in future
Group sequencing, origin Study on Evolution and crossbreeding work etc. can provide valuable reference.To Calanthe cell
The understanding of ploidy is to the screening in advance of the apolegamy of identification, the cross combination of kind, filial generation material, affiliation
Identification etc. all has highly important meaning.
Flow cytometry from 1999 initially as an important technical application into plant research, main application exists
In assessment nuclear content, plant chromosome ploidy and cell cycle analysis are determined.Flow cytometry and other traditional methods
It is quick, convenient and accurate compared to having the characteristics that.The universal of flow cytometry mainly has benefited from relatively simple preparation of samples side
Method, brief method mainly have three steps: (1) shredding Plant tissue samples in suitable buffer, discharge nucleus.(2)
Nucleus is dyed with fluorescent dye, therefore genome is bigger, is dyed also more.(3) machine testing on the nucleus dyed.
By the number of the fluorescent staining amount compared with the plant species of known group size, the Genome Size of sample is obtained.
Most important link is uniformly to crush plant tissue in nucleus lysate in sample preparation.Cell cracking
Buffer should be able to remove remaining cytosolic fraction in complete nucleus, keep stabilization of the nucleus in suspension simultaneously
Prevent agglutination.Cell lysis buffer solution should also be protected and is not degraded, and provided suitable environment and be used to carry out single-minded coring
Learn dyeing, as far as possible negative effect of the reduction cytoplasmic components to dyeing.Since plant tissue is more in structure and chemical composition
Sample is difficult to have a kind of single buffer to be perfectly suitable for all species, part lysate be only applicable to it is a small number of a kind of or
A few plant, this all limits the Ploidy Identification of development plant, estimating for Genome Size.Therefore suitable cracking
Liquid is particularly critical.
Calanthe plant is since its provenance variations is abundant, and widely distributed, form various kinds, genetic background is complicated, in early period
It is found in laboratory research, ideal fluidic cell can not be obtained when using conventional lysate and colouring method processing cell
Art sample, it may appear that miscellaneous peak phenomenon, it is therefore desirable to be improved for the special morphosis of Calanthe plant and physilogical characteristics real
Proved recipe method, so that the sample being finally prepared is suitable for the analysis of flow cytometer.
In consideration of it, the present invention is specifically proposed.
Summary of the invention
In view of the deficiencies of the prior art, the purpose of the present invention is to provide a kind of streaming suitable for Calanthe plant is thin
Calanthe plant can be effectively removed in the preparation method and cell lysis buffer solution of born of the same parents' art sample, the cell lysis buffer solution
Remaining cytosolic fraction in the complete nucleus of object keeps stabilization of the nucleus in suspension and prevents to be aggregated, and provides
Suitable environment is used to carry out single-minded nuclear chemistry dyeing, is effectively reduced negative effect of the cytoplasmic components to dyeing.
Further, in some embodiments of the present invention, the cell lysis buffer solution includes following component are as follows:
MgCl2, Sodium citrate, MOPS, TritonX-100, PVP-10, Tween20.It should be noted that cell cracking is slow
After the completion of fliud flushing is prepared, saved in 4 DEG C of refrigerators.
Further, the configuration of cell lysis buffer solution sequentially includes the following steps:
Step 1: weighing the MgCl of 4.284g2；
Step 2: weighing the Sodium citrate of 5.764g；
Step 3: weighing the MOPS of 4.185g；
Step 4: the above drug being dissolved in the distilled water of 900ml, and the TritonX-100 of 1ml is added；
Step 5: the PVP-10 of 10ml is added；
Step 6: the Tween20 of 5ml is added；
Step 7: being settled to 1000ml, adjust ph to 7.0 with hydrochloric acid, need to illustrate: can after the completion of cell lysis buffer solution configuration
In 4 DEG C of long-term preservations.
Wherein, magnesium chloride is inorganic salts, the integrality of the ionic strength for keeping buffer stable and holding nucleus,
Stability plays the role of preventing nuclear collapse.Citric acid as chelating agent, for combine bivalent cation frequently as it is auxiliary
The enzyme factor prevents from being degraded, and enhances the colouring power and effect of DNA.MOPS is organic buffer liquid, is conducive to the fluorescence dye of DNA
Material dyeing.TritonX-100 is nonionic detergent, is conducive to the release of nucleus and prevents its agglutination.PVP is producing high-molecular
Object is closed, main function is to reduce polyphenols and influence of other secondary metabolites such as tannic acid for fluorescent staining, and
Efficiently reduce the problem that peak figure caused by secondary metabolites is second-rate in Calanthe sample.Tween20 can be improved carefully
The free efficiency of karyon, to increase the content of nucleus in test sample.
Above-mentioned cell lysis buffer solution is effectively gone due to using suitable preparation of reagents, various component synergistic effects
Except remaining cytosolic fraction in the complete nucleus of Calanthe plant, stabilization and prevention of the nucleus in suspension are kept
Agglutination, and suitable environment is provided and is used to carry out single-minded nuclear chemistry dyeing, cytoplasmic components are effectively reduced to the negative of dyeing
It influences.
Another object of the present invention is to provide a kind of flow cytometry sample preparation suitable for Calanthe plant, can obtain
To the suitable Calanthe plant flow cytometry sample for being used for flow cytometry, comprising the following steps:
(1) the blade 50mg for taking Calanthe plant is put into the training for filling the 5.5cm of cell lysis buffer solution of 1-5ml pre-cooling
It supports in ware, blade is cut to fragment with sharp blade, whole process, which operates on ice bag and guarantees that blade is immersed in always, to be split
It solves in liquid, obtains nuclei suspension, stand 5min；
(2) by 400 mesh nylon net filters of nuclei suspension, nucleus filtrate is obtained, 1ml nucleus filtrate is taken to move to once
In property 2ml plastic centrifuge tube；
(3) nucleus filtrate is subjected to centrifugal treating, abandons supernatant and retain precipitating, the lysate that 1ml is added suspends again；
(4) the PI dyestuff of the RnaseA and 50ul of 50ul are added into step (3) suspension, mixes, was stood at 4 DEG C of dark
Night, the nucleus dyed；
(5) nucleus of dyeing is detected and analyzed in flow cytometer.
Further, in embodiments of the invention, the quality of Calanthe tissue sample and cell lysis buffer solution
Volume ratio is 50mg:1ml.Such as the Calanthe vegetable material of 50mg is taken to need the lysate of 1ml.
Further, in centrifugal treating described in step (3), revolving speed 2000-3000rpm, temperature is 4 DEG C, and the time is
5-8min。
Further, the concentration of step (4) the RnaseA enzyme is 1 mgml-1。
Further, the concentration of step (4) the PI dyestuff is 1 mgml-1。
Further, the Calanthe plant includes any one in Chinese Calanthe plant.
Further, in some embodiments in the present invention, which further includes detecting step, after dyeing
Nucleus is obtained to be detected and analyzed in fluidic cell instrument.
The invention has the following advantages:
(1) contain 0.1 %V/V TritonX-100,1%PVP-10,0.5% V/V Tween20 in present invention cracking formula of liquid,
Three is used cooperatively, and can not only achieve the purpose that dissolve cytoplasm and cell membrane, while it is complete to remove Calanthe plant
Nucleus in remaining cytosolic fraction, keep stabilization of the nucleus in suspension and prevent to be aggregated, protection is not degraded,
And suitable environment is provided and is used to carry out single-minded nuclear chemistry dyeing, it is effectively reduced negative effect of the cytoplasmic components to dyeing.
(2) staining procedure in Calanthe plant flow cytometry sample preparation methods is taken and was stood at 4 DEG C of dark
At night, different from the method that traditional short time stands, which can efficiently reduce miscellaneous peak in Calanthe plant examination with computer
Occur, improves accuracy, the Stability and veracity of testing result.In addition, provided by the present invention for preparing Calanthe
The cell lysis buffer solution of plant flow cytometry sample, by the way that suitable preparation of reagents is used, various components act synergistically,
It can be effectively removed remaining cytosolic fraction in the complete nucleus of Calanthe plant, keep nucleus in suspension
Stablize and prevent to be aggregated, and suitable environment is provided and is used to carry out single-minded nuclear chemistry dyeing, is effectively reduced cytoplasmic components pair
The negative effect of dyeing.
Detailed description of the invention
The flow cytometry sample preparation methods institute suitable for Calanthe plant that Fig. 1 provides for the embodiment of the present invention 1
Flow cytometry analysis result of the length prepared away from calanthe flow cytometry sample.
The flow cytometry sample preparation methods institute suitable for Calanthe plant that Fig. 2 provides for the embodiment of the present invention 2
Flow cytometry analysis result of the stick prepared away from calanthe flow cytometry sample.
The flow cytometry sample preparation methods institute suitable for Calanthe plant that Fig. 3 provides for the embodiment of the present invention 3
The flow cytometry analysis result for the fork lip calanthe flow cytometry sample prepared.
The flow cytometry sample preparation methods institute suitable for Calanthe plant that Fig. 4 provides for the embodiment of the present invention 4
The flow cytometric analysis results for the sword-like leave calanthe flow cytometry sample prepared.
The flow cytometry sample preparation methods institute suitable for Calanthe plant that Fig. 5 provides for the embodiment of the present invention 5
The flow cytometric analysis results for the Chinese herbaceous peony calanthe flow cytometry sample prepared.
The flow cytometry sample preparation methods institute suitable for Calanthe plant that Fig. 6 provides for comparative example 1 of the present invention
Flow cytometry analysis result of the length prepared away from calanthe flow cytometry sample.
The flow cytometry sample preparation methods institute suitable for Calanthe plant that Fig. 7 provides for comparative example 2 of the present invention
Flow cytometry analysis result of the length prepared away from calanthe flow cytometry sample.
Specific embodiment
A kind of flow cytometry sample preparation methods suitable for Calanthe plant comprising cleavage step is by calanthe
Platymiscium tissue sample carries out cracking processing, filtration treatment, centrifugal treating, obtains nuclear targeting step to be dyed to described
Nucleus to be dyed carries out dyeing processing, the nucleus dyed.
Configure cell lysis buffer solution:
Step 1: weighing the MgCl of 4.284g2；
Step 2: weighing the Sodium citrate of 5.764g；
Step 3: weighing the MOPS of 4.185g；
Step 4: the above drug being dissolved in the distilled water of 900ml, and the TritonX-100 of 1ml is added；
Step 5: the PVP-10 of 10ml is added；
Step 6: the Tween20 of 5ml is added；
Step 7: being settled to 1000ml, with salt acid for adjusting pH to 7.0, place 4 DEG C of refrigerator pre-coolings.
Embodiment 1
Sample preparation:
(1) it takes length away from calanthe young leaflet tablet 50mg, is put into the culture dish for filling the 5.5cm of cell lysis buffer solution of pre-cooling
In, blade is cut to fragment with sharp blade, whole process operates on ice bag and guarantees that blade is immersed in lysate always
In, nuclei suspension is obtained, 5min is stood.
(2) by 400 mesh nylon net filters of nuclei suspension, nucleus filtrate is obtained, 1ml nucleus filtrate is taken to move to
In disposable 2ml plastic centrifuge tube.
(3) nucleus filtrate is subjected to centrifugal treating 8min in 4 DEG C of revolving speed 2000rpm, abandons supernatant and retain precipitating, is added
The lysate of 1ml suspends again.
(4) first the PI dyestuff of the RnaseA and 50ul of backward suspension addition 50ul, mixing are put being put at 4 DEG C of dark
It sets overnight, the nucleus dyed.
(5) nucleus of dyeing is detected and analyzed in flow cytometer, as a result as shown in Figure 1.
Embodiment 2
Sample preparation:
(1) it takes stick away from calanthe young leaflet tablet 50mg, is put into the culture dish for filling the 5.5cm of cell lysis buffer solution of pre-cooling
In, blade is cut to fragment with sharp blade, whole process operates on ice bag and guarantees that blade is immersed in lysate always
In, nuclei suspension is obtained, 5min is stood.
(2) by 400 mesh nylon net filters of nuclei suspension, nucleus filtrate is obtained, 1ml nucleus filtrate is taken to move to
In disposable 2ml plastic centrifuge tube.
(3) nucleus filtrate is subjected to centrifugal treating 8min in 4 DEG C of revolving speed 2500rpm, abandons supernatant and retain precipitating, is added
The lysate of 1ml suspends again.
(4) first the PI of the RnaseA and 50ul of backward suspension addition 50ul, mixing were placed being put at 4 DEG C of dark
Night, the nucleus dyed.
(5) nucleus of dyeing is detected and analyzed in flow cytometer, as a result as shown in Figure 2.
Embodiment 3
Sample preparation:
(1) fork lip calanthe young leaflet tablet 50mg is taken, the culture dish for filling the 5.5cm of cell lysis buffer solution of pre-cooling is put into
In, blade is cut to fragment with sharp blade, whole process operates on ice bag and guarantees that blade is immersed in lysate always
In, nuclei suspension is obtained, 5min is stood.
(2) by 400 mesh nylon net filters of nuclei suspension, nucleus filtrate is obtained, 1ml nucleus filtrate is taken to move to
In disposable 2ml plastic centrifuge tube.
(3) nucleus filtrate is subjected to centrifugal treating 5min in 4 DEG C of revolving speed 2000rpm, abandons supernatant and retain precipitating, is added
The lysate of 1ml suspends again.
(4) first the PI of the RnaseA and 50ul of backward suspension addition 50ul, mixing were placed being put at 4 DEG C of dark
Night, the nucleus dyed.
(5) nucleus of dyeing is detected and analyzed in flow cytometer, as a result as shown in Figure 3.
Embodiment 4
Sample preparation:
(1) sword-like leave calanthe young leaflet tablet 50mg is taken, the culture dish for filling the 5.5cm of cell lysis buffer solution of pre-cooling is put into
In, blade is cut to fragment with sharp blade, whole process operates on ice bag and guarantees that blade is immersed in lysate always
In, nuclei suspension is obtained, 5min is stood.
(2) by 400 mesh nylon net filters of nuclei suspension, nucleus filtrate is obtained, 1ml nucleus filtrate is taken to move to
In disposable 2ml plastic centrifuge tube.
(3) nucleus filtrate is subjected to centrifugal treating 5min in 4 DEG C of revolving speed 2000rpm, abandons supernatant and retain precipitating, is added
The lysate of 1ml suspends again.
(4) first the PI of the RnaseA and 50ul of backward suspension addition 50ul, mixing were placed being put at 4 DEG C of dark
Night, the nucleus dyed.
(5) nucleus of dyeing is detected and analyzed in flow cytometer, as a result as shown in Figure 4.
Embodiment 5
Sample preparation:
(1) calanthe young leaflet tablet 50mg, is put into the culture dish for filling the 5.5cm of cell lysis buffer solution of pre-cooling before picking up the car
In, blade is cut to fragment with sharp blade, whole process operates on ice bag and guarantees that blade is immersed in lysate always
In, nuclei suspension is obtained, 5min is stood.
(2) by 400 mesh nylon net filters of nuclei suspension, nucleus filtrate is obtained, 1ml nucleus filtrate is taken to move to
In disposable 2ml plastic centrifuge tube.
(3) nucleus filtrate is subjected to centrifugal treating 8min in 4 DEG C of revolving speed 3000rpm, abandons supernatant and retain precipitating, is added
The lysate of 1ml suspends again.
(4) first the PI of the RnaseA and 50ul of backward suspension addition 50ul, mixing were placed being put at 4 DEG C of dark
Night, the nucleus dyed.
(5) nucleus of dyeing is detected and analyzed in flow cytometer, as a result as shown in Figure 5.
By embodiment 1-5 it is found that the cell lysis buffer solution and colouring method that are prepared using the present invention are prepared
Length away from calanthe, stick away from calanthe, fork lip calanthe, sword-like leave calanthe, Chinese herbaceous peony calanthe flow cytometry sample, carrying out
When flow cytometry, the appearance of miscellaneous peak is significantly reduced, improves accuracy, the Stability and veracity of testing result.
Compare case 1
Conventional cracking formula of liquid:
Step 1: weighing the MgCl of 4.2484g2；
Step 2: weighing the Sodium citrate of 8.823g；
Step 3: weighing the MOPS of 4.185g；
Step 4: weighing 0.3722gNa2EDTA；
Step 5: the PVP-10 of 1ml is added；
Step 6: the beta-hydroxy ethyl alcohol of 1.5ml is added；
Step 7: being settled to 200ml, with salt acid for adjusting pH to 7.0, place 4 DEG C of refrigerator pre-coolings.
(1) it takes length away from calanthe young leaflet tablet 50mg, is put into the 5.5cm's for filling the regular growth lysis buffer of pre-cooling
In culture dish, blade is cut to fragment with sharp blade, whole process operates on ice bag and guarantees that blade is immersed in always
In conventional lysate, nuclei suspension is obtained, stands 5min.
(2) by 400 mesh nylon net filters of nuclei suspension, nucleus filtrate is obtained, 1ml nucleus filtrate is taken to move to
In disposable 2ml plastic centrifuge tube.
(3) nucleus filtrate is subjected to centrifugal treating 8min in 4 DEG C of revolving speed 2000rpm, abandons supernatant and retain precipitating, is added
The lysate of 1ml suspends again.
(4) first the PI dyestuff of the RnaseA and 50ul of backward suspension addition 50ul, mixing are put being put at 4 DEG C of dark
It sets overnight, the nucleus dyed.
(5) nucleus of dyeing is detected and analyzed in flow cytometer, as a result as shown in fig. 6, having more miscellaneous
Peak occurs.
Case 1 is compared compared with embodiment it is found that provided by the present invention for preparing Calanthe plant flow cytometry
The cell lysis buffer solution of sample, by the way that suitable preparation of reagents is used, various component synergistic effects can be effectively removed shrimp
Remaining cytosolic fraction in the complete nucleus of ridge epidendrum keeps stabilization and prevention of the nucleus in suspension solidifying
Collection, and suitable environment is provided and is used to carry out single-minded nuclear chemistry dyeing, cytoplasmic components are effectively reduced to the negative shadow of dyeing
It rings.
Compare case 2
Sample preparation:
(1) it takes length away from calanthe young leaflet tablet 50mg, is put into the culture dish for filling the 5.5cm of cell lysis buffer solution of pre-cooling
In, blade is cut to fragment with sharp blade, whole process operates on ice bag and guarantees that blade is immersed in lysate always
In, nuclei suspension is obtained, 5min is stood.
(2) by 400 mesh nylon net filters of nuclei suspension, nucleus filtrate is obtained, 1ml nucleus filtrate is taken to move to
In disposable 2ml plastic centrifuge tube.
(3) nucleus filtrate is subjected to centrifugal treating 8min in 4 DEG C of revolving speed 2000rpm, abandons supernatant and retain precipitating, is added
The lysate of 1ml suspends again.
(4) first the PI dyestuff of the RnaseA and 50ul of backward suspension addition 50ul, mixing are put being put at 4 DEG C of dark
20min is set, the nucleus dyed.
(5) nucleus of dyeing is detected and analyzed in flow cytometer, as a result as shown in fig. 7, having more miscellaneous
Peak occurs.
By the comparison case 2 it is found that taking traditional standing 20min, with 4 DEG C dark at stand overnight (embodiment) phase
Than standing overnight the appearance that can efficiently reduce miscellaneous peak in Calanthe plant examination with computer, improving the accurate of testing result
Property, Stability and veracity.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
Claims (5)
1. a kind of preparation method of the flow cytometry sample suitable for Calanthe plant, which is characterized in that at cracking
Reason and dyeing processing, specifically includes the following steps:
(1) the blade 50mg for taking Calanthe plant is put into the training for filling the 5.5cm of cell lysis buffer solution of 1-5ml pre-cooling
It supports in ware, blade is cut to fragment with sharp blade, whole process, which operates on ice bag and guarantees that blade is immersed in always, to be split
It solves in liquid, obtains nuclei suspension, stand 5min；
(2) by 400 mesh nylon net filters of nuclei suspension, nucleus filtrate is obtained, 1ml nucleus filtrate is taken to move to once
In property 2ml plastic centrifuge tube；
(3) nucleus filtrate is subjected to centrifugal treating, abandons supernatant and retain precipitating, the lysate that 1ml is added suspends again；
(4) the PI dyestuff of the RnaseA and 50ul of 50ul are added into step (3) suspension, mixes, was stood at 4 DEG C of dark
Night, the nucleus dyed；
(5) nucleus of dyeing is detected and analyzed in flow cytometer；
The cell lysis buffer solution used in step (1) described cleavage step includes following component: 45 mmol/L MgCl2, 30
Mmol/L Sodium citrate, 20 mmol/L MOPS, 0.1 %V/V TritonX-100,1%PVP-10,0.5% V/V
7.0,4 DEG C of Tween20, pH preservations.
2. the flow cytometry sample preparation methods according to claim 1 suitable for Calanthe plant, feature exist
In in centrifugal treating described in step (3), revolving speed 2000-3000rpm, temperature is 4 DEG C, time 5-8min.
3. being existed according to claim or the preparation method of the flow cytometry sample suitable for Calanthe plant, feature
In the concentration of step (4) the RnaseA enzyme is 1 mgml-1。
4. the preparation method suitable for the flow cytometry sample of Calanthe plant, feature exist according to claim 1
In the concentration of step (4) the PI dyestuff is 1 mgml-1。
5. the preparation method suitable for the flow cytometry sample of Calanthe plant, feature exist according to claim 1
In the Calanthe plant includes any one in Chinese Calanthe plant.
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