CN109468361B - Method for measuring mononuclear/macrophage capacity of fish natural killer cell killing bacterial infection - Google Patents
Method for measuring mononuclear/macrophage capacity of fish natural killer cell killing bacterial infection Download PDFInfo
- Publication number
- CN109468361B CN109468361B CN201811442073.0A CN201811442073A CN109468361B CN 109468361 B CN109468361 B CN 109468361B CN 201811442073 A CN201811442073 A CN 201811442073A CN 109468361 B CN109468361 B CN 109468361B
- Authority
- CN
- China
- Prior art keywords
- fish
- natural killer
- cells
- macrophages
- mononuclear
- 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.)
- Expired - Fee Related
Links
- 210000002540 macrophage Anatomy 0.000 title claims abstract description 53
- 241000251468 Actinopterygii Species 0.000 title claims abstract description 43
- 210000000822 natural killer cell Anatomy 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 25
- 208000035143 Bacterial infection Diseases 0.000 title description 2
- 208000022362 bacterial infectious disease Diseases 0.000 title description 2
- 230000022534 cell killing Effects 0.000 title description 2
- 210000001616 monocyte Anatomy 0.000 claims abstract description 27
- 241000607471 Edwardsiella tarda Species 0.000 claims abstract description 19
- 239000007853 buffer solution Substances 0.000 claims abstract description 17
- 210000003734 kidney Anatomy 0.000 claims abstract description 16
- 238000005406 washing Methods 0.000 claims abstract description 14
- 108010002350 Interleukin-2 Proteins 0.000 claims abstract description 13
- 241000894006 Bacteria Species 0.000 claims abstract description 12
- 230000002147 killing effect Effects 0.000 claims abstract description 12
- 210000002421 cell wall Anatomy 0.000 claims abstract description 3
- 210000005087 mononuclear cell Anatomy 0.000 claims abstract description 3
- 230000004936 stimulating effect Effects 0.000 claims abstract description 3
- 210000004027 cell Anatomy 0.000 claims description 38
- 239000001963 growth medium Substances 0.000 claims description 22
- 239000007788 liquid Substances 0.000 claims description 12
- 239000002609 medium Substances 0.000 claims description 12
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 11
- 238000012258 culturing Methods 0.000 claims description 11
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 10
- 239000006285 cell suspension Substances 0.000 claims description 9
- 238000005119 centrifugation Methods 0.000 claims description 7
- 238000000926 separation method Methods 0.000 claims description 7
- 210000004698 lymphocyte Anatomy 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 claims description 4
- 229930182566 Gentamicin Natural products 0.000 claims description 4
- 210000000265 leukocyte Anatomy 0.000 claims description 4
- 229920004890 Triton X-100 Polymers 0.000 claims description 3
- 239000013504 Triton X-100 Substances 0.000 claims description 3
- 230000001464 adherent effect Effects 0.000 claims description 3
- 239000003242 anti bacterial agent Substances 0.000 claims description 3
- 229940088710 antibiotic agent Drugs 0.000 claims description 3
- 238000005336 cracking Methods 0.000 claims description 3
- 229960002518 gentamicin Drugs 0.000 claims description 3
- 210000002751 lymph Anatomy 0.000 claims description 3
- 210000005084 renal tissue Anatomy 0.000 claims description 3
- 230000001580 bacterial effect Effects 0.000 claims description 2
- 230000003115 biocidal effect Effects 0.000 claims description 2
- 239000011248 coating agent Substances 0.000 claims description 2
- 238000000576 coating method Methods 0.000 claims description 2
- 230000028744 lysogeny Effects 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000007873 sieving Methods 0.000 claims description 2
- 239000000243 solution Substances 0.000 claims description 2
- 238000011534 incubation Methods 0.000 abstract description 7
- 230000006698 induction Effects 0.000 abstract description 2
- 102000000588 Interleukin-2 Human genes 0.000 description 9
- 241000252230 Ctenopharyngodon idella Species 0.000 description 4
- 239000000872 buffer Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 210000002865 immune cell Anatomy 0.000 description 3
- 210000000987 immune system Anatomy 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 238000007664 blowing Methods 0.000 description 2
- 239000000306 component Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 229940088592 immunologic factor Drugs 0.000 description 1
- 239000000367 immunologic factor Substances 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for measuring the ability of a natural killer cell of fish to kill mononuclear/macrophage infected by bacteria, which comprises the following steps: separating the renal leucocytes of the fish head; preparing fish mononuclear cells/macrophages by a cell wall pasting method; edwardsiella tarda infects monocytes/macrophages; stimulating the fish head kidney leucocyte with IL-2 for 48h, and then washing with sterile PBS buffer solution for 2-3 times to obtain natural killer cells; natural killer cells were co-incubated with Edwardsiella tarda infected monocytes/macrophages, and then plated to determine killing ability based on the number of colonies on the plate. The invention has killing effect on the infected mononuclear/macrophage of Edwardsiella tarda by the fish natural killer cells generated by IL-2 induction, and the killing capability can be judged according to the number of colonies after co-incubation.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for measuring the ability of a natural killer cell of fish to kill mononuclear/macrophage infected by bacteria.
Background
The fish immune system consists of nonspecific immunity and specific immunity. The fish immune system also consists of immune tissues and organs, immune cells and humoral immune factors. There are two types of immune cells: firstly, lymphocytes mainly perform specific immune reaction; the other is phagocytes, which are key components of nonspecific immunity and play an important role in combating microbial infections. The non-specific immune system plays a more important role in the defense of fish against pathogenic invasion. The macrophage is a leukocyte, originates from monocyte, and is mainly used for phagocytizing and digesting cell debris and pathogens in the form of fixed or free cells, and activating immune cells such as lymphocytes and the like to react with the pathogens and prevent the pathogens from diffusing and growing, so the macrophage plays an extremely important role in the immune process of fish. However, the macrophage is infected by bacteria to cause fish to produce adverse symptoms, which affects the survival of the fish, the natural killer cells of the fish can play a role in killing the fish, and how to judge the killing capability of the natural killer cells of the fish is an urgent problem to be solved.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for measuring the capability of the natural killer cells of fishes to kill the monocytes/macrophages infected by the bacteria, which can simply and effectively judge the capability of the natural killer cells to kill the monocytes/macrophages infected by the bacteria.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
a method for measuring the ability of natural killer cells of fishes to kill monocytes/macrophages infected by bacteria comprises the following steps:
(1) separating the renal leucocytes of the fish head;
(2) preparing fish mononuclear cells/macrophages by a cell wall pasting method;
(3) edwardsiella tarda infects monocytes/macrophages;
(4) stimulating the fish head kidney leucocyte with IL-2 with the concentration of 8-12ng/mL for 48h, and then washing with sterile PBS buffer solution for 2-3 times to prepare the fish natural killer cell;
(5) incubating natural killer cells and Edwardsiella tarda infected mononuclear/macrophages for 3.5-4.5h, sucking supernatant, repeatedly washing the cells for 5-6 times by using sterile PBS buffer solution, adding 1% Triton X-100 into the rest cells to lyse the cells for 4-6min, repeatedly blowing and beating the lysed liquid, and uniformly mixing the lysed liquid and the sterile PBS buffer solution;
(6) and (5) coating the uniformly mixed liquid obtained in the step (5) on an LB (lysogeny broth) plate without antibiotics, culturing overnight, counting the number of colonies on the plate, and determining the killing capacity of the fish natural killer cells on the bacterial infected mononuclear/macrophage according to the number of the colonies, wherein the small number of the colonies indicates that the killing capacity is strong.
Further, the specific process in the step (1) is as follows:
first, fish are sacrificed under aseptic conditions, head and kidney are removed with aseptic tools and placed in RPMI1640+10% FBS +1% A+/A+In the medium (2) and (3) washing the head kidney with the medium, and then washing the head kidney with RPMI1640+10% FBS +1% A+/A+Gently squeezing the tissue of the head and kidney to exude cells in the tissue of the head and kidney, collecting cell suspension to a new centrifuge tube, and repeating the step for 3-4 times;
secondly, sieving the cell suspension by a 200-mesh cell sieve, then adding a lymphocyte separation solution, and centrifuging for 25-35min at 20 ℃ under the condition of 2800 r/min;
③ after the centrifugation is finished, sucking the white flocculent cell layer between the culture medium and the lymph separation liquid, mixing the white flocculent cell layer with the sterile PBS buffer solution uniformly, and centrifuging the mixture for 15 to 20min at the temperature of 20 ℃ and under the condition of 2800 r/min;
fourthly, abandoning the supernatant after the centrifugation is finished, and using RPMI1640+10% FBS +1% A+/A+And (4) resuspending the cells in the culture medium to obtain the culture medium.
Further, the specific process in the step (2) is as follows: fish head kidney leukocytes were centrifuged and then treated with DMEM +5% FBS +1% A+/A+The medium (2) is resuspended at 5-10X 106Inoculating each cell/hole into a culture plate, standing for 2-3h at 400 uL/hole, removing the culture medium, adding sterile PBS buffer solution, repeatedly washing for 5-6 times, removing the suspended cells, and leaving the adherent cells as the monocytes/macrophages.
Further, the specific process in the step (3) is as follows: inoculating mononuclear/macrophage into DMEM +5% FBS culture medium without antibiotic, adding Edwardsiella tarda 10 times the amount of mononuclear/macrophage after culturing for 1h, centrifuging at 20 deg.C and 1000g × g for 10min, culturing at 26 deg.C for 40min, absorbing the culture medium, repeatedly washing with sterile PBS buffer solution for 8-10 times, and culturing in DMEM +5% FBS +100ug/mL gentamycin culture medium for 1-2 h.
Further, the concentration of IL-2 in step (4) was 10 ng/mL.
The method for measuring the capability of the natural killer cells of fishes to kill the monocytes/macrophages infected by bacteria has the following beneficial effects:
the invention has killing effect on the infected mononuclear/macrophage of Edwardsiella tarda by the fish natural killer cells generated by IL-2 induction, and the killing capability can be judged according to the number of colonies after co-incubation.
Drawings
FIG. 1 is a flow chart of an experiment according to the present invention;
FIG. 2 shows fish monocytes/macrophages prepared by the adherence method.
FIG. 3 is monocytes/macrophages after Edwardsiella tarda infection.
FIG. 4 is the plate colony results of the control and experimental groups after co-incubation.
FIG. 5 is a histogram of colonies from the control and experimental groups after co-incubation.
Detailed Description
The invention takes grass carp as a research object to carry out experiments, and culture medium components RPMI1640, FBS and double-antibody A used in the experimental process+/A+They were all products from Saimer Feishel scientific Co., Ltd, and the concentration of the sterile PBS buffer was 0.01mol/L and the pH was 7.4. The present invention will be described in detail with reference to examples.
Example 1
A method for measuring the ability of natural killer cells of fishes to kill monocytes/macrophages infected by bacteria comprises the following steps:
1. separating grass carp head kidney leucocyte
(1) The fish were sacrificed under sterile conditions, the head and kidney were removed with a sterile instrument and placed in a chamber containing 3.5mL of RPMI1640+10% FBS +1% A+/A+In a culture dish of the medium, the head kidney was washed 2 times with the medium, and then 5mL of RPMI1640+10% FBS +1% A was added+/A+The head kidney tissue was gently squeezed to exude the cells, the cell suspension was collected into a new centrifuge tube, the procedure was repeated 3 times to obtain a total of 15mL of cell suspension, and then RPMI1640+10% FBS +1% A was added+/A+Medium to 20 mL;
(2) filtering the cell suspension by using a sterile 200-mesh cell sieve, slowly adding the cell suspension into a 50mL centrifuge tube containing lymphocyte separation liquid along the tube wall, centrifuging for 30min at 20 ℃ and 2800r/min, wherein the added cell suspension is 8mL, and the added lymphocyte separation liquid is 8 mL;
(3) after the centrifugation is finished, sucking a white flocculent cell layer between the culture medium and the lymph separation liquid by using a Pasteur pipette, transferring the white flocculent cell layer into a new 50mL centrifuge tube, adding sterile PBS buffer solution with the same volume, washing by light shaking, and centrifuging for 16min at the temperature of 20 ℃ and the speed of 2800 r/min;
(4) discarding the supernatant after the centrifugation in step (3), and adding 5mL of RPMI1640+10% FBS +1% A+/A+The medium resuspended the cells and counted using a hemocytometer.
2. Preparation of grass carp natural killer cells and monocytes/macrophages
Dividing the separated grass carp head kidney white blood cells into two parts, wherein one part is used for preparing natural killer cells, and the other part is used for preparing monocytes/macrophages.
Preparing natural killer cells: will contain 6X 1061.2mL of the culture medium of each cell was added to a 35mm petri dish, followed by stimulation with IL-2 at a concentration of 10ng/mL for 48 hours, and finally washed 2-3 times with sterile PBS buffer.
The control group was supplemented with only 1.2mL of medium, without IL-2.
Preparation of fish monocytes/macrophages: the number of the grooves is 8 multiplied by 107After centrifugation of the cells at 20 ℃ and 2800r/min for 16min, 2mL of DMEM +5% FBS +1% A was added+/A+The medium of (2) is resuspended at 6X 106And (3) inoculating each cell/well into a 24-well culture plate (6 replicates are set in each group of experiment), wherein the volume is 400 uL/well, standing for 2 hours, removing the culture medium, repeatedly washing for 5 times by using 500 uL/well sterile PBS buffer solution, removing the suspension cells, and leaving the adherent cells, namely the monocytes/macrophages (as shown in figure 2).
3. Edwardsiella tarda infection mononuclear/macrophage
(1) Replacing the culture medium of the mononuclear/macrophage with a DMEM and 5% FBS culture medium without antibiotics, and adding 10 times of the amount of the Edwardsiella tarda of the mononuclear/macrophage after culturing for 1h in a 24-hole culture plate;
(2) sealing the 24-well culture plate with sealing membrane, centrifuging at 20 deg.C and 1000g × g for 10min, increasing contact of Edwardsiella tarda with monocyte/macrophage, and culturing at 26 deg.C for 40 min.
The 24-well plate was removed and observed under a microscope, and the results are shown in FIG. 3, and it can be seen from FIG. 3 that Edwardsiella tarda was coated on the surface of monocytes/macrophages.
(3) The culture medium was aspirated, the infected monocytes/macrophages were washed repeatedly with sterile PBS buffer for 8 times, the medium was changed to DMEM +5% FBS +100ug/mL gentamicin, 200 uL/well, and cultured at 26 ℃ for 1h to kill extracellular Edwardsiella tarda.
4. Natural killer cells and Edwardsiella tarda infected monocytes/macrophages co-incubation
(1) Resuspending the prepared natural killer cells in a culture medium of DMEM +5% FBS +100ug/mL gentamicin, counting by a blood counting chamber, inoculating the natural killer cells and infected mononuclear/macrophage in a 24-well culture plate in different proportions, culturing at 200 uL/well, and incubating for 4 h;
(2) sucking the supernatant after 4h, repeatedly washing the cells for 5 times by using sterile PBS buffer solution, adding 1% Triton X-100 into the rest cells at 200 uL/hole, cracking the cells for 5min, obviously observing the cell rupture under a microscope, repeatedly blowing the bottom of a culture dish by using a 100uL pipette, sucking the liquid after cracking at 100 uL/hole, adding the liquid into 1mL of sterile PBS buffer solution, and uniformly mixing by vortex;
(3) 100uL of each sample was inoculated onto antibiotic-free LB plates, incubated overnight at 37 ℃ and then the number of colonies was counted for each plate.
The results of the plate colonies of infected monocytes/macrophages after co-incubation are shown in FIG. 4 and the results of the bar graph are shown in FIG. 5, with IL-2 added to the experimental group and without IL-2 to the control group.
As can be seen from FIGS. 4 and 5, the number of infected mononuclear/macrophage colonies is significantly lower than that of the control group after IL-2 is added, which indicates that the IL-2-induced natural killer cells have killing effect on Edwardsiella tarda infected mononuclear/macrophage colonies, and the killing ability of the Edwardsiella tarda infected mononuclear/macrophage colonies can be determined according to the number of colonies after co-incubation.
Claims (4)
1. A method for measuring the capability of natural killer cells of fishes to kill monocytes/macrophages infected by bacteria is characterized by comprising the following steps:
(1) separating the renal leucocytes of the fish head, which comprises the following specific processes:
taking out the head and kidney of fish with aseptic tool, and adding RPMI1640, 10% FBS and 1% double-antibody A+/A+The medium (A) washes the head kidney 2-3 times, then again in RPMI1640+10% FBS +1% double antibody A+/A+Squeezing the head kidney tissue in the culture medium to exude cells in the head kidney tissue, collecting cell suspension, and repeating the step for 3-4 times;
secondly, sieving the cell suspension by a 200-mesh cell sieve, then adding a lymphocyte separation solution, and centrifuging for 25-35min at 20 ℃ under the condition of 2800 r/min;
③ after the centrifugation is finished, sucking the white flocculent cell layer between the culture medium and the lymph separation liquid, mixing the white flocculent cell layer with the sterile PBS buffer solution uniformly, and centrifuging the mixture for 15 to 20min at the temperature of 20 ℃ and under the condition of 2800 r/min;
fourthly, abandoning the supernatant after the centrifugation is finished, and using RPMI1640+10% FBS +1% double-antibody A+/A+Suspending cells by culture medium to obtain;
(2) preparing fish mononuclear cells/macrophages by a cell wall pasting method;
(3) edwardsiella tarda infects monocytes/macrophages;
(4) stimulating the fish head kidney leucocyte with IL-2 with the concentration of 8-12ng/mL for 48h, and then washing the cell with sterile PBS buffer solution to prepare a fish natural killer cell;
(5) incubating natural killer cells and Edwardsiella tarda infected mononuclear/macrophages for 3.5-4.5h, sucking supernatant, repeatedly washing the cells by using sterile PBS buffer solution, then cracking the cells by using 1% Triton X-100, then adding the sterile PBS buffer solution into the cracked liquid, and uniformly mixing;
(6) and (5) coating the uniformly mixed liquid obtained in the step (5) on an LB (lysogeny broth) plate without antibiotics, culturing overnight, counting the number of colonies on the plate, and measuring the killing capacity of the fish natural killer cells on the bacterial infected mononuclear/macrophage according to the number of the colonies.
2. The method for determining the ability of fish natural killer cells to kill monocytes/macrophages infected with bacteria according to claim 1, wherein the specific process in step (2) is: centrifuging fish head kidney leukocyte, and treating with DMEM, 5% FBS and 1% double antibody A+/A+The medium (2) is resuspended at 5-10X 106Inoculating each cell/hole into a culture plate, standing for 2-3h, removing the culture medium, adding sterile PBS buffer solution, repeatedly washing for 5-6 times, removing the suspended cells, and leaving the adherent cells as the monocytes/macrophages.
3. The method for determining the ability of fish natural killer cells to kill monocytes/macrophages infected with bacteria according to claim 1, wherein the specific process in step (3) is: inoculating mononuclear/macrophage into DMEM +5% FBS culture medium without antibiotic, adding Edwardsiella tarda 10 times the amount of mononuclear/macrophage after culturing for 1h, then centrifuging for 10min at 20 ℃ under 1000g, finally culturing for 40min at 26 ℃, absorbing the culture medium, repeatedly washing for 8-10 times by using sterile PBS buffer solution, and culturing for 1-2h in DMEM +5% FBS +100ug/mL gentamicin culture medium.
4. The method for measuring the ability of fish natural killer cells to kill monocytes/macrophages infected with bacteria according to claim 1, wherein the concentration of IL-2 in step (4) is 10 ng/mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811442073.0A CN109468361B (en) | 2018-11-29 | 2018-11-29 | Method for measuring mononuclear/macrophage capacity of fish natural killer cell killing bacterial infection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811442073.0A CN109468361B (en) | 2018-11-29 | 2018-11-29 | Method for measuring mononuclear/macrophage capacity of fish natural killer cell killing bacterial infection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109468361A CN109468361A (en) | 2019-03-15 |
| CN109468361B true CN109468361B (en) | 2021-11-02 |
Family
ID=65673379
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811442073.0A Expired - Fee Related CN109468361B (en) | 2018-11-29 | 2018-11-29 | Method for measuring mononuclear/macrophage capacity of fish natural killer cell killing bacterial infection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109468361B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110196219B (en) * | 2019-05-16 | 2022-06-03 | 扬州大学 | Chicken spleen CD8 detection based on flow cytometry+Method for specific killing of T cells |
| CN111676265B (en) * | 2020-05-29 | 2021-06-15 | 威海市立医院 | Method for detecting phagocytic capacity of neutrophil |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101892241A (en) * | 2010-07-09 | 2010-11-24 | 电子科技大学 | Grass carp interleukin 1 beta gene and protein and recombinant expression method thereof |
| CN102041260A (en) * | 2011-01-04 | 2011-05-04 | 中山大学 | Grouper interleukin EcIL-1beta gene, coded protein, and preparation method and application thereof |
| CN103468636A (en) * | 2013-09-26 | 2013-12-25 | 上海大学 | Method for extracting and identifying carp primary macrophage |
| EP2479277B1 (en) * | 2004-07-22 | 2015-09-02 | Five Prime Therapeutics, Inc. | Use of MGD-CSF in a method of treatment of Alzheimer's disease. |
| CN106011061A (en) * | 2016-08-04 | 2016-10-12 | 广东省第二人民医院 | In-vitro large-scale amplification method of natural killer cells |
| CN107254439A (en) * | 2009-12-29 | 2017-10-17 | 加米达细胞有限公司 | Strengthen the method for NK propagation and activity |
| CN108822202A (en) * | 2018-02-07 | 2018-11-16 | 电子科技大学 | A kind of 21 recombinant protein of leucocytes of grass carp interleukin and preparation method thereof |
-
2018
- 2018-11-29 CN CN201811442073.0A patent/CN109468361B/en not_active Expired - Fee Related
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2479277B1 (en) * | 2004-07-22 | 2015-09-02 | Five Prime Therapeutics, Inc. | Use of MGD-CSF in a method of treatment of Alzheimer's disease. |
| CN107254439A (en) * | 2009-12-29 | 2017-10-17 | 加米达细胞有限公司 | Strengthen the method for NK propagation and activity |
| CN101892241A (en) * | 2010-07-09 | 2010-11-24 | 电子科技大学 | Grass carp interleukin 1 beta gene and protein and recombinant expression method thereof |
| CN102041260A (en) * | 2011-01-04 | 2011-05-04 | 中山大学 | Grouper interleukin EcIL-1beta gene, coded protein, and preparation method and application thereof |
| CN103468636A (en) * | 2013-09-26 | 2013-12-25 | 上海大学 | Method for extracting and identifying carp primary macrophage |
| CN106011061A (en) * | 2016-08-04 | 2016-10-12 | 广东省第二人民医院 | In-vitro large-scale amplification method of natural killer cells |
| CN108822202A (en) * | 2018-02-07 | 2018-11-16 | 电子科技大学 | A kind of 21 recombinant protein of leucocytes of grass carp interleukin and preparation method thereof |
Non-Patent Citations (5)
| Title |
|---|
| "IL-2活化人NK细胞的作用机理";邬于川等;《免疫学杂志》;20041130;第20卷(第6期);第487页 * |
| "In vitro characterization of grass carp (Ctenopharyngodon idella) IL-26 in regulating inflammatory factors";Xingyang Qiu等;《Fish & Shellfish Immunology》;20170508;第66卷;第148-155页 * |
| "Monocyte/Macrophage: NK Cell Cooperation—Old Tools for New Functions";Elzbieta Walajtys-Rode等;《Results Probl Cell Differ》;20171231;第62卷;第73-145页 * |
| "RIP2对大肠杆菌的抗菌作用及其机制研究";席琼;《中国优秀硕士学位论文全文数据库医药卫生科技辑》;20111015(第10期);第1页方法部分 * |
| "草鱼头肾免疫细胞组成和数量变化";卢全章;《动物学研究》;19981231;第19卷(第1期);摘要 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109468361A (en) | 2019-03-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102268405B (en) | Method for auto NK (Natural Killer) cell in-vitro activation and amplification culture and special culture medium thereof | |
| CN109468361B (en) | Method for measuring mononuclear/macrophage capacity of fish natural killer cell killing bacterial infection | |
| CN103756963A (en) | Method used for in vitro proliferation of NK cells | |
| CN111394310A (en) | Enhanced NK cell for strong immunoregulation and strong killing of tumor cells and virus infected cells, and preparation method and kit thereof | |
| CN104498434A (en) | Preparation method of large number of dendritic cells and obtained dendritic cells | |
| CN107034189A (en) | A kind of candidate stem cell cultural method | |
| CN105112371A (en) | Preparation method for DC-CIK cells originated from umbilical cord blood mononuclear cells and preparation | |
| CN112608896A (en) | NK cell culture method and application thereof | |
| CN112251405A (en) | Method for efficiently inducing and amplifying NK cells in vitro | |
| US20230123462A1 (en) | In vitro proliferation medium, in vitro culture kit and in vitro proliferation culture method of umbilical cord blood (ucb)-derived natural killer (nk) cells | |
| CN116948986A (en) | Antibacterial tolerance multivalent vibrio alginolyticus phage VAP230304 and application thereof | |
| WO2016201658A1 (en) | Method for preparing tumor specific ctls | |
| CN104762261A (en) | Tumor infiltrating lymphocytes separation method | |
| CN111548994A (en) | Cell culture medium and method for culturing NK cells by using same | |
| CN110862962A (en) | Method for culturing and amplifying NK cells in vitro by using gallic acid | |
| CN111154721B (en) | NK cell amplification method | |
| CN105535940A (en) | Preparation method of Vgamma9Vdelta2T cell preparation for treating multiple myeloma | |
| CN105462925A (en) | Preparing method for high-toxicity human Vgamma9Vdelta2 T cells induced by PD-1 antibody | |
| CN114480279A (en) | Efficient separation culture technology for human blood immune cells CD4T | |
| CN109576220B (en) | Method for stimulating NK cell amplification by combining inactivated feeder cells with cytokines | |
| CN107779435A (en) | A kind of co-cultivation supernatant containing autologous CIK cell and its application | |
| CN114686432B (en) | Efficient amplification culture system for neutrophils and application thereof | |
| CN107779434A (en) | The experimental method of efficient amplification Human peripheral blood NK cells | |
| Oestmann et al. | A method for producing microbe-free Amyloodinium ocellatum (Brown) with Percoll® | |
| CN111088227A (en) | Cell separation culture solution and T cell separation culture method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20211102 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |