CN110806374B - Binding Buffer for detecting Annexin V apoptosis and preparation method thereof - Google Patents
Binding Buffer for detecting Annexin V apoptosis and preparation method thereof Download PDFInfo
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- CN110806374B CN110806374B CN201910931952.8A CN201910931952A CN110806374B CN 110806374 B CN110806374 B CN 110806374B CN 201910931952 A CN201910931952 A CN 201910931952A CN 110806374 B CN110806374 B CN 110806374B
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- 239000012148 binding buffer Substances 0.000 title claims abstract description 36
- 230000006907 apoptotic process Effects 0.000 title claims abstract description 22
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- 108090000672 Annexin A5 Proteins 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title abstract description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 14
- 229940098773 bovine serum albumin Drugs 0.000 claims description 14
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 238000003756 stirring Methods 0.000 claims description 12
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 10
- 239000007995 HEPES buffer Substances 0.000 claims description 10
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 8
- 239000012498 ultrapure water Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 5
- 238000005303 weighing Methods 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims 1
- 238000010186 staining Methods 0.000 abstract description 18
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 29
- 102000000412 Annexin Human genes 0.000 description 12
- 108050008874 Annexin Proteins 0.000 description 12
- 210000000170 cell membrane Anatomy 0.000 description 7
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 description 5
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- YXHLJMWYDTXDHS-IRFLANFNSA-N 7-aminoactinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=C(N)C=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 YXHLJMWYDTXDHS-IRFLANFNSA-N 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 108700012813 7-aminoactinomycin D Proteins 0.000 description 3
- 238000003782 apoptosis assay Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- 230000005522 programmed cell death Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 206010063746 Accidental death Diseases 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 108091093105 Nuclear DNA Proteins 0.000 description 1
- 239000000980 acid dye Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 230000004900 autophagic degradation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 230000010428 chromatin condensation Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000004987 nonapoptotic effect Effects 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 102000036213 phospholipid binding proteins Human genes 0.000 description 1
- 108091011000 phospholipid binding proteins Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000021419 recognition of apoptotic cell Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
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- 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
- G01N15/1404—Handling flow, e.g. hydrodynamic focusing
-
- 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
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- 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
- G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
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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
- 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
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- 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
- G01N2015/1006—Investigating individual particles for cytology
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Abstract
The invention discloses a Binding Buffer for detecting Annexin V apoptosis and a preparation method thereof, which solve the problems of unobvious cell grouping, low staining index and the like; compared with the commercial Binding Buffer on the market, the reagent can obviously improve the staining index and make the grouping between cells more obvious.
Description
Technical Field
The invention relates to Binding Buffer for detecting Annexin V apoptosis and a preparation method thereof.
Background
Apoptosis (Apoptosis), also known as Programmed Cell Death (PCD), is an active, orderly Death of cells controlled by genes, is an important way for the organism to regulate and maintain the relative balance of the organism, and is closely related to various disease pathologies. The main forms of PCD are apoptosis, autophagy, and pyro-death, among others, and are distinguished from necrosis and accidental death. As an important component in the cell life cycle, the apoptosis process is accompanied by the change of cell morphology and the activation, expression and regulation of a series of genes. The earliest change of apoptosis of cells occurs on cell membranes, and Phosphatidylserine (PS) turns from the inner layer of the cell membrane to the outer layer of the cell membrane, so that the PS is exposed on the outer surface of the cell membrane, and the change is earlier than the apoptosis phenomena of cell shrinkage, chromatin condensation, nuclear DNA fragmentation and the like.
Annexin V is a human endogenous protein, belongs to a calcium ion-dependent phospholipid binding protein family, and has an anticoagulation effect. Annexin V has high affinity with PS and therefore binds to PS exposed outside the cell membrane during early apoptosis. Annexin V labels fluorescein FITC, PE, APC and the like, and the apoptosis phenomenon is detected by using a flow cytometer and the like, so that the Annexin V labels fluorescein FITC, PE, APC and the like can be used as one of sensitive indexes for detecting early apoptosis of cells. This is currently the accepted method of sensitive, efficient and specific detection of apoptotic cells.
Propidium Iodide (PI) and 7-amino actinomycin (7-AAD) are commonly used nucleic acid dyes that do not penetrate the intact cell membrane, but late in apoptosis or dead cells, PI and 7-AAD are able to penetrate the cell membrane to stain the nucleus. Therefore, the use of Annexin V in combination with PI or 7-AAD allows differentiation between cells at different stages.
Most of the Annexin V apoptosis detection methods utilize a flow cytometer, the flow cytometer emits laser to excite a fluorescent dye through a built-in laser, emits emission light with another specific wavelength, and performs detection analysis through a detector. The Annexin V labels such as micromolecular dyes FITC or protein PE, APC and the like, and because the labeling ratio of the Annexin V to the fluorescent dye, separation and purification after labeling and the like can cause that grouping after cell dyeing is not obvious, and the dyeing index is low. During the cell treatment process, the non-specific binding of the non-apoptotic cells after treatment to the fluorescent dye may cause the staining of the cells to be less distinct. Annexin V is used as a sensitive index for detecting early apoptosis of cells, and false positive or false negative can be generated in an analysis result because cell grouping after staining is not obvious and cells in different periods cannot be distinguished obviously.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a Binding Buffer for Annexin V apoptosis detection and a preparation method thereof, aiming at solving the problems of unobvious cell grouping, low staining index and the like.
In order to achieve the purpose, the invention provides the following technical scheme: a Binding Buffer for detecting Annexin V apoptosis comprises the following components and formula:
HEPES，50mM-60mM；
sodium chloride, 700mM-1000 mM;
calcium chloride, dihydrate, 12.5mM-15 mM;
BSA，1％-5％；
Proclin95，0.1％-0.2％；
the balance of water
The pH value of the Binding Buffer is 7.0-7.4;
preferably, the Binding Buffer comprises the following components in percentage by weight:
HEPES，50mM；
sodium chloride, 700 mM;
calcium chloride, dihydrate, 12.5 mM;
BSA，5％；
Proclin95，0.2％；
the balance of water
The pH value of the Binding Buffer is 7.4.
The preparation method comprises the following steps:
the method comprises the following steps: accurately weighing HEPES, sodium chloride, calcium chloride, dihydrate and BSA (bovine serum albumin) into a beaker, adding a proper amount of ultrapure water, stirring and uniformly mixing, and fully dissolving;
step two: accurately measuring Proclin950, adding into the solution, uniformly stirring, standing for half an hour, and adjusting the pH value to 7.4 by using a 10N NaOH solution;
step three: after the pH is adjusted, adding ultrapure water into the beaker by using a measuring cylinder, fixing the volume to the prepared volume, and stirring and uniformly mixing;
step four: filtering, and storing at low temperature.
Preferably, filtration through a 0.22 μm filter is carried out.
Preferably, the low temperature is 4 ℃.
The invention has the beneficial effects that: the problems of unobvious cell grouping, low staining index and the like are solved; compared with commercial Binding Buffer on the market, the reagent can obviously improve the staining index and make the grouping between cells more obvious.
Drawings
FIG. 1 shows the flow detection result of commercially available Binding Buffer treated cell staining Annexin V-PE of the present invention, wherein:
FIG. 1-1 is a flow scattergram of commercially available Binding Buffer, Annexin V-PE dyed;
FIG. 1-2 is a commercially available Binding Buffer, Annexin V-PE stained flow histogram (staining index 6.8);
FIGS. 1-3 are flow scattergrams of Binding Buffer, Annexin V-PE of the present invention after staining;
FIGS. 1-4 are flow histograms (staining index 14.9) after staining Binding Buffer, Annexin V-PE of the present invention.
FIG. 2 is a flow-type detection result of Annexin V-APC after commercial and Binding Buffer treatment in the market, wherein:
FIG. 2-1 is a flow scattergram of commercially available Binding Buffer, Annexin V-APC after staining;
FIG. 2-2 is a commercially available Binding Buffer, Annexin V-APC stained flow histogram (staining index 6.8);
FIG. 2-3 is a flow scattergram of Binding Buffer, Annexin V-APC of the present invention after staining;
FIGS. 2-4 are flow histograms (staining index 14.9) of Binding Buffer, Annexin V-APC staining of the present invention
Detailed Description
Example 1
A Binding Buffer for detecting Annexin V apoptosis comprises the following components and formula:
HEPES，50mM；
sodium chloride, 700 mM;
calcium chloride, dihydrate, 12.5 mM;
BSA，5％；
Proclin95，0.2％；
the balance of water
The Binding buffer pH value is 7.4;
the method comprises the following steps:
1. accurately weighing HEPES, sodium chloride, calcium chloride, dihydrate and BSA (bovine serum albumin) into a beaker, adding a proper amount of ultrapure water, stirring and uniformly mixing, and fully dissolving;
2. accurately measuring Proclin950, adding into the solution, uniformly stirring, standing for half an hour, and adjusting the pH value to 7.4 by using a 10N NaOH solution;
3. after the pH is adjusted, adding ultrapure water into the beaker by using a measuring cylinder, fixing the volume to the prepared volume, and stirring and uniformly mixing;
4. after filtration through a 0.22 μm filter, the cells were stored at 4 ℃.
As shown in figure 1 of the drawings, in which,
5X 105-1X 106 Eca109 cells were collected, washed twice with pre-cooled PBS by centrifugation, and the supernatant was discarded.
The cells were resuspended in 500. mu.l of positive control solution and incubated on ice for 30 minutes.
The cells were washed with pre-cooled PBS by centrifugation and the supernatant was discarded.
Adding a proper amount of precooled 1 × Binding Buffer for resuspension, adding the same amount of untreated living cells, and mixing uniformly. Add precooling 1 × Binding Buffer to make up to 1ml, divide the tube into two equal portions, one is blank control tube, one is singly dyed tube.
Add 5. mu.l Annexin V-PE to the single staining tube and incubate for 10 min at room temperature in the dark.
Example 2
A Binding Buffer for detecting Annexin V apoptosis comprises the following components and formula:
HEPES，60mM；
sodium chloride, 1000 mM;
calcium chloride, dihydrate, 15 mM;
BSA，1％；
Proclin95，0.2％；
the balance of water
The Binding buffer pH value is 7.4;
the method comprises the following steps:
1. accurately weighing HEPES, sodium chloride, calcium chloride, dihydrate and BSA (bovine serum albumin) into a beaker, adding a proper amount of ultrapure water, stirring and uniformly mixing, and fully dissolving;
2. accurately measuring Proclin950, adding into the solution, uniformly stirring, standing for half an hour, and adjusting the pH value to 7.4 by using a 10N NaOH solution;
3. after the pH is adjusted, adding ultrapure water into the beaker by using a measuring cylinder, fixing the volume to the prepared volume, and stirring and uniformly mixing;
4. after filtration through a 0.22 μm filter, the cells were stored at 4 ℃.
As shown in figure 2 of the drawings, in which,
5X 105-1X 106 jurkat cells were collected, washed twice by centrifugation in pre-cooled PBS and the supernatant discarded.
The cells were resuspended in 500. mu.l of positive control solution and incubated on ice for 30 minutes.
The cells were washed with pre-cooled PBS by centrifugation and the supernatant was discarded.
Adding a proper amount of precooled 1 × Binding Buffer for resuspension, adding the same amount of untreated living cells, and mixing uniformly. Add precooling 1 × Binding Buffer to make up to 1ml, divide the tube into two equal portions, one is blank control tube, one is singly dyed tube.
Adding 5 mul Annexin V-APC into a single dyeing tube, and incubating for 10 minutes at room temperature in the dark
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (5)
1. A Binding Buffer for detecting Annexin V apoptosis is characterized by comprising the following components and formula:
HEPES，50mM-60mM；
sodium chloride, 700mM-1000 mM;
calcium chloride, dihydrate, 12.5mM-15 mM;
BSA，1％-5％；
Proclin95，0.1％-0.2％；
the balance of water
The pH value of the Binding Buffer is 7.0-7.4.
2. The Binding Buffer for detecting Annexin V apoptosis of claim 1, wherein the Binding Buffer comprises the following components in percentage by weight:
HEPES，50mM；
sodium chloride, 700 mM;
calcium chloride, dihydrate, 12.5 mM;
BSA，5％；
Proclin95，0.2％；
the balance of water
The pH value of the Binding Buffer is 7.4.
3. The method for preparing Binding Buffer according to claim 1, comprising the steps of:
the method comprises the following steps: accurately weighing HEPES, sodium chloride, calcium chloride, dihydrate and BSA (bovine serum albumin) into a beaker, adding a proper amount of ultrapure water, stirring and uniformly mixing, and fully dissolving;
step two: accurately measuring Proclin950, adding into the solution, uniformly stirring, standing for half an hour, and adjusting the pH value to 7.4 by using a 10N NaOH solution;
step three: after the pH is adjusted, adding ultrapure water into the beaker by using a measuring cylinder, fixing the volume to the prepared volume, and stirring and uniformly mixing;
step four: filtering, and storing at low temperature.
4. The method of claim 3, wherein the Binding Buffer is filtered through a 0.22 μm filter.
5. The method of claim 3, wherein the cooling temperature of the fourth step is 4 ℃.
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2019
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