CN103712967A - Method for detecting activity of NK (Natural Killer) cell - Google Patents
Method for detecting activity of NK (Natural Killer) cell Download PDFInfo
- Publication number
- CN103712967A CN103712967A CN201410005008.7A CN201410005008A CN103712967A CN 103712967 A CN103712967 A CN 103712967A CN 201410005008 A CN201410005008 A CN 201410005008A CN 103712967 A CN103712967 A CN 103712967A
- Authority
- CN
- China
- Prior art keywords
- cell
- target cell
- fluorescent dye
- fluorescent
- target
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 32
- 230000000694 effects Effects 0.000 title abstract description 20
- 210000004027 cell Anatomy 0.000 claims abstract description 228
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 58
- 210000000822 natural killer cell Anatomy 0.000 claims abstract description 30
- 238000002156 mixing Methods 0.000 claims abstract description 24
- 238000005516 engineering process Methods 0.000 claims abstract description 12
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 12
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 12
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 12
- 238000001890 transfection Methods 0.000 claims abstract description 12
- 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 claims abstract description 11
- 239000000980 acid dye Substances 0.000 claims abstract description 9
- 102000034287 fluorescent proteins Human genes 0.000 claims abstract description 5
- 108091006047 fluorescent proteins Proteins 0.000 claims abstract description 5
- MURGITYSBWUQTI-UHFFFAOYSA-N fluorescin Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC=C(O)C=C2OC2=CC(O)=CC=C21 MURGITYSBWUQTI-UHFFFAOYSA-N 0.000 claims description 52
- 230000006907 apoptotic process Effects 0.000 claims description 48
- 238000001514 detection method Methods 0.000 claims description 34
- 206010027336 Menstruation delayed Diseases 0.000 claims description 22
- 239000007788 liquid Substances 0.000 claims description 22
- 108010043121 Green Fluorescent Proteins Proteins 0.000 claims description 16
- 238000004043 dyeing Methods 0.000 claims description 15
- 102000004144 Green Fluorescent Proteins Human genes 0.000 claims description 14
- 239000005090 green fluorescent protein Substances 0.000 claims description 14
- 241000700605 Viruses Species 0.000 claims description 12
- 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 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- DPKHZNPWBDQZCN-UHFFFAOYSA-N acridine orange free base Chemical compound C1=CC(N(C)C)=CC2=NC3=CC(N(C)C)=CC=C3C=C21 DPKHZNPWBDQZCN-UHFFFAOYSA-N 0.000 claims description 4
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 claims description 4
- 108010082025 cyan fluorescent protein Proteins 0.000 claims description 4
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 claims description 4
- 229960005542 ethidium bromide Drugs 0.000 claims description 4
- 238000012856 packing Methods 0.000 claims description 4
- 150000003904 phospholipids Chemical class 0.000 claims description 4
- 239000013600 plasmid vector Substances 0.000 claims description 4
- 108010054624 red fluorescent protein Proteins 0.000 claims description 4
- 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 claims description 3
- 108700012813 7-aminoactinomycin D Proteins 0.000 claims description 3
- 108010004729 Phycoerythrin Proteins 0.000 claims description 3
- 108010004469 allophycocyanin Proteins 0.000 claims description 3
- 108091005948 blue fluorescent proteins Proteins 0.000 claims description 3
- 238000010276 construction Methods 0.000 claims description 3
- 208000035250 cutaneous malignant susceptibility to 1 melanoma Diseases 0.000 claims description 3
- 201000001441 melanoma Diseases 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 230000001640 apoptogenic effect Effects 0.000 abstract description 8
- 102000036213 phospholipid binding proteins Human genes 0.000 abstract 1
- 108091011000 phospholipid binding proteins Proteins 0.000 abstract 1
- 210000000170 cell membrane Anatomy 0.000 description 23
- 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 10
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 description 10
- 210000004369 blood Anatomy 0.000 description 8
- 239000008280 blood Substances 0.000 description 8
- 239000012636 effector Substances 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 238000000684 flow cytometry Methods 0.000 description 6
- 102000000412 Annexin Human genes 0.000 description 5
- 108050008874 Annexin Proteins 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 102000004121 Annexin A5 Human genes 0.000 description 4
- 108090000672 Annexin A5 Proteins 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000012148 binding buffer Substances 0.000 description 3
- 230000022534 cell killing Effects 0.000 description 3
- 238000004737 colorimetric analysis Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000012447 hatching Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000007405 data analysis Methods 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 239000003822 epoxy resin Substances 0.000 description 2
- 230000002147 killing effect Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 229920000647 polyepoxide Polymers 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000003146 transient transfection Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- QGZCUOLOTMJILH-UHFFFAOYSA-N 2h-tetrazol-2-ium;bromide Chemical compound [Br-].C1=N[NH+]=NN1 QGZCUOLOTMJILH-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 239000012099 Alexa Fluor family Substances 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010023126 Jaundice Diseases 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 241000736199 Paeonia Species 0.000 description 1
- 235000006484 Paeonia officinalis Nutrition 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010598 annexinV-PE /7AAD assay Methods 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 238000000295 emission spectrum Methods 0.000 description 1
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 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 1
- 238000012921 fluorescence analysis Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000009610 hypersensitivity Effects 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000003153 stable transfection Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a method for detecting the activity of an NK (Natural Killer) cell. The method comprises the following steps: specifically expressing a fluorescent protein through a target cell by virtue of a transfection technology to obtain a transfected target cell; co-incubating the NK cell to be detected and the transfected target cell to obtain first mixed cell sap; adding a first fluorescent dye to the first mixed cell sap, and incubating to obtain second mixed cell sap; adding a second fluorescent dye to the second mixed cell sap, and mixing to obtain third mixed cell sap; detecting the third mixed cell sap by a flow cytometer to obtain the cell count ratio of live target cells to early apoptotic target cells to finally apoptotic target cells to dead target cells, wherein the first fluorescent dye is a fluorescein-labeled phospholipid binding protein, the second fluorescent dye is a nucleic acid dye, and the emission wavelengths of the fluorescent protein, the first fluorescent dye and the second fluorescent dye are different from each other. The method for detecting the activity of the NK cell, which is disclosed by the invention, can integrally reflect the activity of the NK cell.
Description
Technical field
The present invention relates to cell detection field, in particular to the method that detects NK cytoactive.
Background technology
Natural killer cell (natural killer cell, NK) be effector cell important in body natural immune system, not only with antitumor, viral infection resisting is relevant, and has immunoloregulation function, participates in some cases the generation of hypersensitivity and autoimmune disease.The detection of NK cytoactive is an important process in medical research or clinical research.
For the detection of NK cytoactive, successively work out multiple detection method at present, for example
51cr method for releasing, lactic dehydrogenase (LDH) method for releasing, tetrazolium bromide (MTT) colourimetry and flow cytometry etc.Wherein, flow cytometry, owing to having no radioactivity pollute, stability advantages of higher, therefore obtains paying close attention to more and more widely.
In correlation technique, flow cytometry detects the conventional means of NK cytoactive and is: first by transient transfection, make the specific expressed green fluorescent protein of target cell (GFP); Then hatch altogether target cell and NK cell; After hatching a period of time, re-use propidium iodide (PI) mark dead cell; The fluorescence intensity of the fluorescence intensity of the target cell finally detect living and dead target cell, obtains the activity of NK cell according to both ratio.It is a kind of nucleotide fluorescent dye that this detection method mainly exists following shortcoming: PI, the non-viable apoptotic cell and the dead cell that only can labeled cell film lose integrality, therefore above-mentioned detection method cannot reflect the ratio of NK cell to early apoptosis of cells (now cell membrane integrity still exists) due in target cell kill process.
Summary of the invention
The object of the present invention is to provide the method that detects NK cytoactive, to solve the above problems.
The method that detects NK cytoactive is provided in an embodiment of the present invention, has comprised the following steps:
Utilize rotaring dyeing technology to make the specific expressed fluorescin of target cell, obtain transfection target cell;
NK cell to be measured and transfection target cell are hatched altogether, obtain the first cell mixing liquid;
In the first cell mixing liquid, add the first fluorescent dye, hatch, obtain the second cell mixing liquid;
In the second cell mixing liquid, add the second fluorescent dye, mix, obtain the 3rd cell mixing liquid;
Utilize flow cytometer to detect the 3rd cell mixing liquid, obtain the cell number ratio of the target cell that lives, the cell number ratio of the target cell of early apoptosis, late period apoptosis the cell number ratio of target cell and the cell number ratio of dead target cell;
Wherein, the first fluorescent dye is fluorescein-labeled phospholipids incorporate albumen, and the second fluorescent dye is nucleic acid dye, and the emission wavelength of fluorescin, the first fluorescent dye and the second fluorescent dye is different.
Further, rotaring dyeing technology is:
The slow virus plasmid vector of construction expression fluorescin, and packing forms slow virus;
With slow virus, infect target cell, genes of interest is incorporated in the genome of target cell.
Further, fluorescin is with lower a kind of: green fluorescent protein, cyan fluorescent protein, red fluorescent protein, orange fluorescent protein, yellow fluorescence protein, blue fluorescent protein or purple fluorescence albumen.
Further, in the first fluorescent dye, fluorescein is with lower a kind of: phycoerythrin, fluorescein isothiocynate, enhancement mode fluorescin, allophycocyanin.
Further, the second fluorescent dye is with lower a kind of: 7-aminoactinomycin D, propidium iodide, ethidium bromide, acridine orange, 4,6-diamidino-2-phenylindone or Hext.
Further, target cell is lower a kind of: K-562 cell, HeLa cell or malignant melanoma cell system.
The method of the detection NK cytoactive of the above embodiment of the present invention has realized the object of the ratio of the target cell that simultaneously detects different conditions (i.e. the target cell of target cell alive, early apoptosis, late period apoptosis and dead target cell) by two methods of dying, can reflect more all sidedly the activity of NK cell.
The concrete principle of above detection method is: first by rotaring dyeing technology, make the specific expressed fluorescin of target cell, thereby realize the object of specific recognition target cell, the fluorescence that even sends above-mentioned fluorescin is target cell.Secondly, NK cell to be measured and transfection target cell are hatched altogether, specific binding occurs and cause target cell apoptosis.In the first cell mixing liquid, add the first fluorescent dye again, make its specifically with phosphatidylserine (PS) combination that is exposed to cell membrane outside; In the second cell mixing liquid, add the second fluorescent dye again, make the apoptosis of its specific marker eucaryotic cell structure integrality destruction and the nucleus of dead cell.Due to apoptotic early stage, PS can be turned to the surface of cell membrane from the inner side of cell membrane, be exposed in extracellular environment, so the target cell of early apoptosis can significantly be dyeed by the first fluorescent dye.And in apoptotic late period, although the destructiveness of cell membrane is larger, but also there is a certain amount of cell membrane turning up, therefore, late period, the target cell of apoptosis also can be by the first fluorescent dyeing, and the structure of target cell membrane of apoptosis is imperfect due to late period, therefore, the target cell membrane that the second fluorescent dye (nucleic acid dye) can see through apoptosis in late period enters in nucleus nucleus is dyeed.And the cell membrane of dead target cell is destroyed substantially completely, therefore almost cannot be by the first fluorescent dyeing, but the nucleic acid substances of dead target cell still exists, and therefore can be by the second fluorescent dyeing.
As can be seen here, by above step, reach following object: the fluorescin of take in step 101 as green fluorescent protein be example, all target cells send green fluorescence; The target cell of early apoptosis sends the fluorescence of green fluorescence and the first fluorescent dye, and the second fluorescent dye is refused to dye, and does not send the fluorescence of the second fluorescent dye; Late period, the target cell of apoptosis can send the fluorescence of green fluorescence, the first fluorescent dye and the fluorescence of the second fluorescent dye; Dead target cell can send the fluorescence of green fluorescence and the second fluorescent dye.Finally, luminous characteristics in conjunction with the target cell of above four kinds of states, utilize flow cytometer to detect, can obtain the cell number ratio of target cell alive, the cell number ratio of the target cell of early apoptosis, late period apoptosis the cell number ratio of target cell and the cell number ratio of dead target cell.
Accompanying drawing explanation
Fig. 1 shows the image of GFP-K562 cell under 10 * 10 power microscopes;
Fig. 2 shows the image of GFP-K562 cell under 10 * 40 power microscopes;
Fig. 3 shows the scatter diagram of the GFP-K562 cell GFP expression rate of cells were tested by flow cytometry;
Fig. 4 shows the peak value figure of the GFP-K562 cell GFP expression rate of cells were tested by flow cytometry;
Fig. 5 shows the simple GFP-K562 natural apoptosis ratio distribution plan that flow cytometer detects;
The active result of the NK cell that Fig. 6 shows the normal person that flow cytometer detects to GFP-K562 cell;
The active result of the NK cell that Fig. 7 shows the patient that flow cytometer detects to GFP-K562 cell.
Embodiment
Below by specific embodiment, also by reference to the accompanying drawings the present invention is described in further detail.
Embodiment mono-
A method that detects NK cytoactive, comprises the following steps:
Step 101: utilize rotaring dyeing technology method to make the specific expressed fluorescin of target cell, obtain transfection target cell.
Step 102: NK cell to be measured and transfection target cell are hatched altogether, obtain the first cell mixing liquid.
Step 103: add the first fluorescent dye in the first cell mixing liquid, hatch, obtain the second cell mixing liquid.
Step 104: add the second fluorescent dye in the second cell mixing liquid, mix, obtain the 3rd cell mixing liquid.
Step 105: utilize flow cytometer to detect the 3rd cell mixing liquid, obtain the cell number ratio of the target cell that lives, the cell number ratio of the target cell of early apoptosis, late period apoptosis the cell number ratio of target cell and the cell number ratio of dead target cell.
Wherein, the first fluorescent dye is fluorescein-labeled phospholipids incorporate albumen, and the second fluorescent dye is nucleic acid dye, and the emission wavelength of fluorescin, the first fluorescent dye and the second fluorescent dye is different.
Above detection method has realized the object of the ratio of the target cell that simultaneously detects different conditions target cell and the dead target cell of apoptosis (i.e. the target cell of target cell alive, early apoptosis, late period) by two methods of dying, can reflect more all sidedly the activity of NK cell.
The concrete principle of above detection method is: first by cell transfecting technology, make the specific expressed fluorescin of target cell, thereby realize the object of specific recognition target cell, the fluorescence that even sends above-mentioned fluorescin is target cell.Secondly, NK cell to be measured and transfection target cell are hatched altogether, specific binding occurs and cause target cell apoptosis.In the first cell mixing liquid, add the first fluorescent dye again, make its specifically with phosphatidylserine (PS) combination that is exposed to cell membrane outside; In the second cell mixing liquid, add the second fluorescent dye again, make the apoptosis of its specific marker eucaryotic cell structure integrality destruction and the nucleus of dead cell.Due to apoptotic early stage, PS can be turned to the surface of cell membrane from the inner side of cell membrane, be exposed in extracellular environment, so the target cell of early apoptosis can significantly be dyeed by the first fluorescent dye.And in apoptotic late period, although the destructiveness of cell membrane is larger, but also there is a certain amount of cell membrane turning up, therefore, late period, the target cell of apoptosis also can be by the first fluorescent dyeing, and the structure of target cell membrane of apoptosis is imperfect due to late period, therefore, the target cell membrane that the second fluorescent dye (nucleic acid dye) can see through apoptosis in late period enters in nucleus nucleus is dyeed.And the cell membrane of dead target cell is destroyed substantially completely, therefore almost cannot be by the first fluorescent dyeing, but the nucleic acid substances of dead target cell still exists, and therefore can be by the second fluorescent dyeing.
As can be seen here, by above step, reach following object: the fluorescin of take in step 101 as green fluorescent protein be example, all target cells send green fluorescence; The target cell of early apoptosis sends the fluorescence of green fluorescence and the first fluorescent dye, and the second fluorescent dye is refused to dye, and does not send the fluorescence of the second fluorescent dye; Late period, the target cell of apoptosis can send the fluorescence of green fluorescence, the first fluorescent dye and the fluorescence of the second fluorescent dye; Dead target cell can send the fluorescence of green fluorescence and the second fluorescent dye.Finally, in conjunction with the luminous characteristics of the target cell of above four kinds of states, utilize flow cytometer to detect, can obtain the cell number ratio of the target cell that lives, the cell number ratio of the target cell of early apoptosis, late period apoptosis cell number ratio and the cell number ratio of dead target cell.
In addition, above detection method also has the following advantages:
By cell transfecting, make the target cell itself can specific expressed fluorescin, thereby saved the miscellaneous process that needs to carry out in advance cell dyeing or antibody labeling before each experiment, also avoided the inaccurate problem of unspecific staining result, avoided hatching altogether the problem of front labels targets impact cell target cell function simultaneously.
Because the detection method sensitivity of the present embodiment improves, therefore, in detecting blood during NK cell active, required blood volume greatly reduces, and through evidence, detection method of the present invention is during for detection of NK cell in blood active, blood drawing amount be conventional art (
51cr method for releasing, LDH method for releasing, MTT colourimetry) 25%.
The detection method of the present embodiment does not need radiomaterial, therefore, does not have radioactive contamination.
Detection time is short: the detection duration of the detection method of the present embodiment is about 2-4h, and need 18-24h the detection time of conventional art (LDH method for releasing, MTT colourimetry etc.), and as can be seen here, greatly shorten the detection time of the present embodiment.
In sum, the detection method of the present embodiment has the plurality of advantages such as detection is comprehensive, accuracy is high, highly sensitive, detection efficiency is high, sample aequum is little.
In the detection method of above embodiment, the reaction conditions of each step or reagent classification, addition and incubation time used can be made advantageous embodiment, to improve technique effect.Specific as follows.
In step 101, cell transfecting technology can adopt the means of the cytotostatic transfection of lentivirus mediated, to avoid transient transfection (to refer to that foreign gene enters after target cell, be present on free carrier, unconformability is in the genome of target cell) after target cell through the genes of interest fragment loss that repeatedly goes down to posterity, cause not expressing the problem of GFP.The concrete grammar of stable transfection is: the slow virus plasmid vector of construction expression fluorescin packing form slow virus; With above-mentioned slow virus, infect target cell, genes of interest is incorporated in the genome of target cell.And, fluorescin wherein can adopt the fluorescin of multiple color, such as green fluorescent protein (GFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), orange fluorescent protein, yellow fluorescence protein (YFP), blue fluorescent protein or purple fluorescence albumen etc.In addition, cell transfecting also can adopt other rotaring dyeing technology, the cell transfecting method of different technologies such as physics mediation, chemistry mediation and other biological mediation.
In step 103, the first fluorescent dye need meet specific marker early apoptosis and late period apoptosis the condition of target cell, i.e. fluorescein-labeled phospholipids incorporate albumen (Annexin V).Because phosphatidylserine (PS) is normally at the inner side of Cell membrane lipids bilayer, but apoptotic early stage, PS can be turned to the surface of cell membrane from the inner side of cell membrane, be exposed in extracellular environment, and Annexin V can be with PS high-affinity, be combined specifically.Annexin V is carried out fluorescein-labelled, can by early apoptosis and late period apoptosis cell dyeing.Wherein, the fluorescein of Annexin V mark is multiple, phycoerythrin (PE for example, send out yellow fluorescence), fluorescein isothiocynate (FITC, jaundice green fluorescence), enhancement mode fluorescin (EGFP, green-emitting fluorescence), allophycocyanin (APC sends out blue-fluorescence) and Ai Likesi series fluorescent dye (Alexa Fluor) etc.In order to distinguish the fluorescence of fluorescin, the first fluorescent dye and the second fluorescent dye, also need ask the wavelength of fluorescence of three kinds of material transmittings different.
In step 104, the second fluorescent dye will meet the condition of specific marker apoptosis in late period and dead target cell, nucleic acid dye can not see through complete cell membrane, but the cell membrane integrity of apoptosis middle and advanced stage cell and dead cell is destroyed, can permeate through cell membranes and make that nucleus is red to be dyed, thereby nucleic acid dye can specific marker apoptosis in late period and dead target cell.Wherein, available nucleic acid dye has multiple, for example 7-aminoactinomycin D (7AAD, send out peony fluorescence), PI(send out red fluorescence), ethidium bromide (EB), acridine orange (AO), 4,6-diamidino-2-phenylindone (DAPI) or Hext (Hoechst) etc.Wherein 7AAD has similar fluorescent characteristic to PI, but the emission spectrum of 7AAD is narrow compared with PI, less to the interference of other sense channels, in multicolor fluorescence analysis, is therefore the best substitute of PI, can combine use with Annexin V-PE, and accuracy in detection is higher.
In addition, fluorescin, the first fluorescent dye and the second fluorescent dye related in step 101,103 and 104 are meeting under the prerequisite of testing conditions, can adopt many kinds of substance to combine, for example fluorescin is that green fluorescent protein, the first fluorescent dye are the combination that Annexin V-PE, the second fluorescent dye are 7AAD, or other combination (herein not repeating).
In the detection method of the present embodiment, target cell used can adopt the multiple target cell of NK cell energy specific recognition, for example, and K-562 cell, HeLa cell or malignant melanoma cell system.Wherein, because K-562 cell is the natural target cell of NK cell, NK cell is sensitiveer to it, therefore preferably adopts K-562 cell.When adopting K-562 cell, in the practical operation of step 102, NK cell to be measured and transfection target cell (effect/target cell) altogether incubation time all can observe comparatively stable NK cell to target cell lethal effect in various degree within the scope of 1～6h, 2～4h more preferably wherein, reason is that testing result is more excellent and be beneficial to completing of testing in this incubation time section, and continue to extend incubation time, testing result does not continue to increase thereupon.In addition, effector cell's to be measured concentration range can select 5 * 10
5cells/mL～1 * 10
7cells/mL, effect/target proportional range can be selected 5:1～40:1, can adopt the arbitrary value in above-mentioned scope equally in actual testing process.Wherein, preferred effector cell's to be measured concentration is 5 * 10
6cells/mL, effect/target ratio is 10:1, under this alternative condition, testing result data are relatively more excellent, required relatively less blood drawing amount, and make full use of to a greater extent reagent, (the too low or too low meeting of effect/target cell ratio of effector cell's concentration causes data less than normal, causes normal person and clinical samples difference not obvious save to detect the consumption of reagent; Effector cell's excessive concentration or effect/target cell ratio are too high by increasing the required blood drawing amount that detects, and increase the consumption that detects reagent simultaneously).In addition, hatch altogether required temperature, CO
2or other nutrient solution all can adopt the condition in routine techniques, for example 37 ℃, 5%CO
2deng.
Embodiment bis-
The testing sample behaviour limosis vein blood that the present embodiment is used, the detection method of NK cytoactive is specific as follows.
Note: the condition such as the reaction time of the proportioning between the concentration of related solution, each reagent, each step, temperature hereinafter; in real work; can adjust as one sees fit or further optimize, and the present invention's value that the protection domain of above parameter is not limited to hereinafter adopted.
1, the foundation of GFP-K562 cell line (being transfection target cell)
K562 cell line is the natural target cell of NK cells of human beings, first build the slow virus plasmid vector that carries GFP genes of interest, and carry out slow virus packing, then with above-mentioned slow virus, infect K562 cell, by resistance culture base, screen, the group that picking is formed by unicellular amplification cultivates, further filter out the cell line that GFP genes of interest and K562 cellular genome are integrated, and carry out real time fluorescent quantitative nucleic acid amplification detection system (qPCR) and verify, finally obtain the K562 stable cell lines (hereinafter referred is GFP-K562 cell) of 1 strain stably express GFP, as shown in Figures 1 to 4.
2, the preparation of effector cell, target cell
Get people's limosis vein blood 2mL, be placed in ethylenediamine tetraacetic acid (EDTA) anti-freezing sterile test tube, phosphate buffer (PBS) dilutes 1 times of slow being added on lymphocyte separation medium afterwards, 2000r/min, 20min centrifuging mononuclearcell, PBS washes 2 times, with making cell concentration containing the RPMI-1640 nutrient solution of 10% hyclone, is 5 * 10
6/ mL PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC) suspension is standby.The GFP-K562 cell of cultivation is washed 2 times with PBS, with trypan blue, detected cell survival rate more than 95%, then with RPMI-1640 nutrient solution, cell concentration is adjusted into 5 * 10
5cells/mL is standby.
3, effector cell and target cell hatch altogether
Get each 100 μ L of above-mentioned effector cell PBMC suspension and target cell GFP-K562 cell suspension and mix in epoxy resin (EP) pipe, effect/target cell concentration ratio is 10:1, puts into 37 ℃, 5%CO
2in incubator, hatch altogether 2h.
4, Flow cytometry NK cell killing activity
Cell suspension after hatching is collected in to flow cytometer detector tube, microcentrifuge 1500r/min, 5min are centrifugal, abandon nutrient culture media, with cold PBS, be connected respectively damping fluid (Binding Buffer) washed cell 1 time with 1X, cell is suspended in 100 μ L1X Binding Buffer, and concentration is approximately 1～5 * 10
6cells/mL, add 5 μ L Annexin V-PE, after mixing gently, in room temperature lucifuge, hatch 10-15 minute, with after 1X Binding Buffer washing with 200 μ L re-suspended cells, add 5 μ L7AAD to mix gently rear room temperature lucifuge and hatch 3-5 minute, carry out flow cytometer detection.Establish respectively that simple GFP-K562 cell Annexin V-PE is mono-to be dyed and the mono-adjusting of dying hole voltage and compensation while making two-parametric analysis of 7AAD.Using the two holes of dying of simple GFP-K562 cell Annexin V-PE/7AAD as the contrast of target cell natural apoptosis background, background contrasts as shown in Figure 5.
5, data analysis
Take respectively GFP and lateral angle scattering SSC makes two-dimentional scatter diagram as coordinate transverse axis and the longitudinal axis, and GFP-K562 cell is established to door, and 10,000 GFP-K562 cells of every pipe sample acquisition carry out data analysis.Take respectively Annexin V-PE and 7AAD makes two-dimentional scatter diagram as coordinate transverse axis and the longitudinal axis, analyzes living cells in the GFP-K562 cell obtaining, early apoptosis, late period apoptosis and dead cell ratio (as shown in Fig. 5,6,7) separately.After effect, target cell acting in conjunction, the apoptosis ratio of target cell has reflected the killing activity of NK cell.NK cell killing activity (%)=(the natural apoptosis ratio of the apoptosis ratio of sample aperture GFP-K562 cell-simple GFP-K562 target cell) * 100%.
Take Fig. 5 as example, the %=5.93% of the total natural apoptosis ratio (%) of simple GFP-K562 cell=(100-94.07).Take Fig. 6 as example, and normal person effect/target cell hatches the %=11.57% of target cell apoptosis ratio (%) after 2h=(100-82.50-5.93) altogether, and the normal person's that surveyed the total killing activity of NK cell is 11.57%.Take Fig. 7 as example, and patient effect/target cell hatches target cell apoptosis ratio (%)=(100-88.11-5.93)=5.96% after 2h altogether, and the patient's that surveys NK cell killing activity is 5.96%.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (6)
1. a method that detects NK cytoactive, is characterized in that, comprises the following steps:
Utilize rotaring dyeing technology to make the specific expressed fluorescin of target cell, obtain transfection target cell;
NK cell to be measured and described transfection target cell are hatched altogether, obtain the first cell mixing liquid;
In described the first cell mixing liquid, add the first fluorescent dye, hatch, obtain the second cell mixing liquid;
In described the second cell mixing liquid, add the second fluorescent dye, mix, obtain the 3rd cell mixing liquid;
Utilize flow cytometer to detect described the 3rd cell mixing liquid, obtain the cell number ratio of the target cell that lives, the cell number ratio of the target cell of early apoptosis, late period apoptosis the cell number ratio of target cell and the cell number ratio of dead target cell;
Wherein, described the first fluorescent dye is fluorescein-labeled phospholipids incorporate albumen, and described the second fluorescent dye is nucleic acid dye, and the emission wavelength of described fluorescin, described the first fluorescent dye and described the second fluorescent dye is different.
2. the method for detection NK cytoactive according to claim 1, is characterized in that, described rotaring dyeing technology is:
The slow virus plasmid vector of construction expression fluorescin, and packing forms slow virus;
With described slow virus, infect target cell, genes of interest is incorporated in the genome of target cell.
3. the method for detection according to claim 1 NK cytoactive, it is characterized in that, described fluorescin is with lower a kind of: green fluorescent protein, cyan fluorescent protein, red fluorescent protein, orange fluorescent protein, yellow fluorescence protein, blue fluorescent protein or purple fluorescence albumen.
4. the method for detection according to claim 1 NK cytoactive, is characterized in that, in described the first fluorescent dye, described fluorescein is with lower a kind of: phycoerythrin, fluorescein isothiocynate, enhancement mode fluorescin, allophycocyanin.
5. the method for detection according to claim 1 NK cytoactive, it is characterized in that, described the second fluorescent dye is with lower a kind of: 7-aminoactinomycin D, propidium iodide, ethidium bromide, acridine orange, 4,6-diamidino-2-phenylindone or Hext.
6. the method for detection according to claim 1 NK cytoactive, is characterized in that, described target cell be with lower a kind of: K-562 cell, HeLa cell or malignant melanoma cell are.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410005008.7A CN103712967B (en) | 2013-12-27 | 2014-01-06 | Method for detecting activity of NK (Natural Killer) cell |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310741395 | 2013-12-27 | ||
| CN201310741395.6 | 2013-12-27 | ||
| CN201410005008.7A CN103712967B (en) | 2013-12-27 | 2014-01-06 | Method for detecting activity of NK (Natural Killer) cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103712967A true CN103712967A (en) | 2014-04-09 |
| CN103712967B CN103712967B (en) | 2014-12-03 |
Family
ID=50406113
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410005008.7A Active CN103712967B (en) | 2013-12-27 | 2014-01-06 | Method for detecting activity of NK (Natural Killer) cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103712967B (en) |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107367455A (en) * | 2017-07-10 | 2017-11-21 | 中国科学院合肥物质科学研究院 | A kind of measuring method of tumour cell tolerance pro apoptotic protein matter molecule amount absolute quantitation |
| CN108333152A (en) * | 2017-01-19 | 2018-07-27 | 中国科学院烟台海岸带研究所 | Biotinylated antibody sensor and preparation method thereof based on genetic recombination phycocyanin MAC and graphene oxide quantum dot |
| CN109477838A (en) * | 2016-05-24 | 2019-03-15 | 蔚山大学校产学协力团 | Active inspection method using the NK cell of receptor synergistic activity and the diagnostic method with the disease of the activity relationships of NK cell using it |
| CN110018143A (en) * | 2019-03-29 | 2019-07-16 | 深圳大学 | The method for detecting dinoflagellate Apoptosis |
| CN110068529A (en) * | 2019-05-05 | 2019-07-30 | 中国科学院深圳先进技术研究院 | A kind of characterizing method of outer vesica |
| CN110231275A (en) * | 2019-05-20 | 2019-09-13 | 西安交通大学 | A method of based on Flow cytometry NK cell killing activity |
| CN110542675A (en) * | 2019-09-30 | 2019-12-06 | 广州市锐博生物科技有限公司 | Apoptosis detection method and apoptosis detection kit |
| CN111073947A (en) * | 2018-10-19 | 2020-04-28 | 三峡大学 | Apoptosis detection kit and detection method and application thereof |
| CN112813133A (en) * | 2021-01-29 | 2021-05-18 | 上海睿钰生物科技有限公司 | Method and system for detecting cell killing efficacy and application thereof |
| CN113155712A (en) * | 2021-05-17 | 2021-07-23 | 上海轩锋生物科技有限公司 | Method for detecting cell activity through flow cytometry |
| CN114774467A (en) * | 2022-04-08 | 2022-07-22 | 华中科技大学 | Preparation method of multiple fluorescence labeling living cell sample |
| WO2022252104A1 (en) * | 2021-06-01 | 2022-12-08 | 张慧敏 | Staining method for live-cell imaging |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080254480A1 (en) * | 2007-04-11 | 2008-10-16 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Microcytoxicity assay by pre-labeling target cells |
| CN102741695A (en) * | 2010-02-11 | 2012-10-17 | 霍夫曼-拉罗奇有限公司 | 3D ADCC NK FACS assay |
-
2014
- 2014-01-06 CN CN201410005008.7A patent/CN103712967B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080254480A1 (en) * | 2007-04-11 | 2008-10-16 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Microcytoxicity assay by pre-labeling target cells |
| CN102741695A (en) * | 2010-02-11 | 2012-10-17 | 霍夫曼-拉罗奇有限公司 | 3D ADCC NK FACS assay |
Non-Patent Citations (2)
| Title |
|---|
| 张俊等: "利用流式细胞仪检测人外周血NK细胞毒性方法的建立", 《实用临床医药杂志》 * |
| 王向玲: "蛋白酶体抑制剂对自然杀伤细胞活性和功能影响的研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109477838A (en) * | 2016-05-24 | 2019-03-15 | 蔚山大学校产学协力团 | Active inspection method using the NK cell of receptor synergistic activity and the diagnostic method with the disease of the activity relationships of NK cell using it |
| CN109477838B (en) * | 2016-05-24 | 2022-06-07 | 蔚山大学校产学协力团 | Method for examining activity of NK cell utilizing receptor synergistic activity and diagnostic method using same |
| CN108333152A (en) * | 2017-01-19 | 2018-07-27 | 中国科学院烟台海岸带研究所 | Biotinylated antibody sensor and preparation method thereof based on genetic recombination phycocyanin MAC and graphene oxide quantum dot |
| CN108333152B (en) * | 2017-01-19 | 2020-12-22 | 中国科学院烟台海岸带研究所 | Biotinylated antibody sensor based on gene recombinant phycocyanin MAC and graphene oxide quantum dots and preparation method thereof |
| CN107367455A (en) * | 2017-07-10 | 2017-11-21 | 中国科学院合肥物质科学研究院 | A kind of measuring method of tumour cell tolerance pro apoptotic protein matter molecule amount absolute quantitation |
| CN107367455B (en) * | 2017-07-10 | 2020-10-16 | 中国科学院合肥物质科学研究院 | Method for measuring absolute quantification of number of tumor cell tolerance pro-apoptosis protein molecules |
| CN111073947A (en) * | 2018-10-19 | 2020-04-28 | 三峡大学 | Apoptosis detection kit and detection method and application thereof |
| CN110018143A (en) * | 2019-03-29 | 2019-07-16 | 深圳大学 | The method for detecting dinoflagellate Apoptosis |
| CN110018143B (en) * | 2019-03-29 | 2022-04-05 | 深圳大学 | Method for detecting dinoflagellate apoptosis |
| CN110068529A (en) * | 2019-05-05 | 2019-07-30 | 中国科学院深圳先进技术研究院 | A kind of characterizing method of outer vesica |
| CN110068529B (en) * | 2019-05-05 | 2023-06-23 | 中国科学院深圳先进技术研究院 | Characterization method of outer vesicle |
| CN110231275A (en) * | 2019-05-20 | 2019-09-13 | 西安交通大学 | A method of based on Flow cytometry NK cell killing activity |
| CN110542675A (en) * | 2019-09-30 | 2019-12-06 | 广州市锐博生物科技有限公司 | Apoptosis detection method and apoptosis detection kit |
| CN110542675B (en) * | 2019-09-30 | 2021-12-14 | 广州市锐博生物科技有限公司 | Apoptosis detection method and apoptosis detection kit |
| CN112813133A (en) * | 2021-01-29 | 2021-05-18 | 上海睿钰生物科技有限公司 | Method and system for detecting cell killing efficacy and application thereof |
| CN112813133B (en) * | 2021-01-29 | 2022-07-15 | 上海睿钰生物科技有限公司 | Method and system for detecting cell killing efficacy and application thereof |
| CN113155712A (en) * | 2021-05-17 | 2021-07-23 | 上海轩锋生物科技有限公司 | Method for detecting cell activity through flow cytometry |
| WO2022252104A1 (en) * | 2021-06-01 | 2022-12-08 | 张慧敏 | Staining method for live-cell imaging |
| CN114774467A (en) * | 2022-04-08 | 2022-07-22 | 华中科技大学 | Preparation method of multiple fluorescence labeling living cell sample |
| CN114774467B (en) * | 2022-04-08 | 2024-03-29 | 华中科技大学 | Preparation method of multiple fluorescent marked living cell sample |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103712967B (en) | 2014-12-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103712967B (en) | Method for detecting activity of NK (Natural Killer) cell | |
| Adan et al. | Flow cytometry: basic principles and applications | |
| Caron et al. | Assessment of bacterial viability status by flow cytometry and single cell sorting | |
| Chan et al. | Rapid image-based cytometry for comparison of fluorescent viability staining methods | |
| Lian et al. | Flow cytometric analysis of nanoscale biological particles and organelles | |
| Yang et al. | Development of an ultrasensitive dual-channel flow cytometer for the individual analysis of nanosized particles and biomolecules | |
| Palková et al. | Single‐cell analysis of yeast, mammalian cells, and fungal spores with a microfluidic pressure‐driven chip‐based system | |
| US20100221769A1 (en) | Electroporative flow cytometry | |
| CN110231275A (en) | A method of based on Flow cytometry NK cell killing activity | |
| CN110018143B (en) | Method for detecting dinoflagellate apoptosis | |
| CN102947266A (en) | Method of analysing a cell or other biological material containing a nucleic acid | |
| Balke et al. | Visualizing oxidative cellular stress induced by nanoparticles in the subcytotoxic range using fluorescence lifetime imaging | |
| CN104198357A (en) | Application of flow cytometry method to vaccine production real-time monitoring | |
| Lanigan et al. | Real time visualization of cancer cell death, survival and proliferation using fluorochrome-transfected cells in an IncuCyte® imaging system | |
| Dashkova et al. | Microalgal cytometric analysis in the presence of endogenous autofluorescent pigments | |
| CA2321203A1 (en) | Selective cell analysis | |
| El‐Hajjar et al. | A guide to flow cytometry: components, basic principles, experimental design, and cancer research applications | |
| FI92076B (en) | Binding of potentially sensitive substances specific to lymphocytes | |
| CN103189495A (en) | Multiple phase flow system for detecting and isolating substances | |
| Kwok et al. | Laser particle barcoding for multi-pass high-dimensional flow cytometry | |
| Bantly et al. | CellVue® Claret, a new far-red dye, facilitates polychromatic assessment of immune cell proliferation | |
| CN115266664B (en) | Method for evaluating in-vitro killing efficacy of immune cell therapeutic preparation and application thereof | |
| CN104515758B (en) | Promote the quantitative detecting method of cancer activity and the screening technique of carcinogenic promoting agent | |
| Chantzoura et al. | Flow cytometry | |
| Lekshmi et al. | A real‐time image‐based approach to distinguish and discriminate apoptosis from necrosis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20181105 Address after: 101109 Beijing Chaoyang District Beijing Shun Tung Street 6 hospital 26 Building 1 stories 101, 2 layer 201, 3 layer 301, 4 layer 401. Patentee after: BEIJING JINYU MEDICAL EXAMINATION LABORATORY Co.,Ltd. Address before: 100050 Yongan Road, Xuanwu District, Beijing 95 Patentee before: BEIJING FRIENDSHIP HOSPITAL, CAPITAL MEDICAL University |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20191128 Address after: 510000 room 706, No. 10, helix 3 Road, international bio Island, Huangpu District, Guangzhou, Guangdong Province Patentee after: Guangzhou jinbilai Biotechnology Co.,Ltd. Address before: 101109 Beijing Chaoyang District Beijing Shun Tung Street 6 hospital 26 Building 1 stories 101, 2 layer 201, 3 layer 301, 4 layer 401. Patentee before: BEIJING JINYU MEDICAL EXAMINATION LABORATORY Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200311 Address after: Room 406, room 101, floor 1, building 55, yard 5, Yujing East Road, Tongzhou District, Beijing 100000 Patentee after: BEIJING BEIKEWEI BIOLOTY TECHNOLOGY Co.,Ltd. Address before: 510000 room 706, No. 10, helix 3 Road, international bio Island, Huangpu District, Guangzhou, Guangdong Province Patentee before: Guangzhou jinbilai Biotechnology Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200929 Address after: 100000 No. 95, Yongan Road, Beijing, Xicheng District Patentee after: Wang Zhao Address before: Room 406, room 101, floor 1, building 55, yard 5, Yujing East Road, Tongzhou District, Beijing 100000 Patentee before: BEIJING BEIKEWEI BIOLOTY TECHNOLOGY Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20231114 Address after: 301700 room 106, building G6, Changyuan road international enterprise community, Wuqing Development Zone, Wuqing District, Tianjin Patentee after: Becoway (Tianjin) Biotechnology Co.,Ltd. Address before: 100000 Yongan Road, Xicheng District, Beijing 95 Patentee before: Wang Zhao |