CN111254112A - Preparation and identification kit for mouse bone marrow macrophages and application thereof - Google Patents
Preparation and identification kit for mouse bone marrow macrophages and application thereof Download PDFInfo
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Images
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70546—Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM
- G01N2333/70553—Integrin beta2-subunit-containing molecules, e.g. CD11, CD18
Abstract
The invention provides a preparation and identification kit of mouse bone marrow macrophages and application thereof, wherein the kit comprises: the kit comprises a kit body, wherein a Phosphate Buffer Solution (PBS) reagent bottle, a streptomycin antibiotic reagent bottle, an erythrocyte lysate reagent bottle, a cell culture medium reagent bottle, a fetal bovine serum reagent bottle, a mouse fibroblast L929 culture supernatant reagent bottle, a CD11b antibody reagent bottle, a bacterial strain bottle for phagocytosis identification and an instruction book are filled in the kit body. The mouse bone marrow macrophage preparation and identification kit is simple and convenient to operate, integrates reagents required by bone marrow macrophage preparation and identification experiments, and effectively improves the preparation efficiency and the survival rate of the culture of the bone marrow-derived macrophages.
Description
Technical Field
The invention relates to the technical field of cells, in particular to a preparation and identification experimental technology of mouse bone marrow macrophages.
Background
Mouse bone marrow-derived macrophages are primary macrophages obtained by in vitro differentiation of undifferentiated bone marrow cells in the presence of macrophage colony stimulating factor (M-CSF) or L929 cell culture supernatant (containing macrophage colony stimulating factor). Primary macrophages can be obtained in relatively high yield, can be stored frozen, and can be obtained from transgenic mouse strains, so that the primary macrophages are primary macrophages widely used for in vitro research in immunology and cell biology.
Macrophages were obtained in 1884 by Russian zoologists
For the first time, it was found that leukocytes, which are an important type of leukocyte in the immune system, have the function of phagocytizing and digesting cell debris, foreign microorganisms, cancer cells, and any other substance that does not have a cell-specific protein of a surface health body. In addition to phagocytosis, they play a key role in non-specific defenses (innate immunity) and also help to initiate specific defense mechanisms (adaptive immunity) by recruiting other immune cells such as lymphocytes. Macrophages are a key component of innate and adaptive immune responses and, due to their strong phagocytic pathogenic microbial activity, they are the first line of defense against foreign invaders. Macrophages are widely distributed throughout the body and are found in the lymphoid organs, liver, lung, gastrointestinal tract, central nervous system, bone and skin. Due to their redistribution, participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells capable of recognizing changes in the microenvironment and maintaining tissue homeostasis. Many pathogens have evolved mechanisms that survive, replicate, infect, and propagate throughout the body of humans and animals by macrophages as trojan horses. Recent scientific interest in macrophages has been renewed by evolutionary, genetic, and biochemical aspects of host-pathogen interactions.
Isolation of resident macrophages from mouse tissues has low productivity and complicated procedures, which have restricted many studies of functional macrophages. The differentiation of bone marrow cells from precursor cells into macrophages by culturing the cells in vitro with appropriate growth factors is currently a widely accepted practice in the industry. However, the preparation of the macrophage derived from the mouse bone marrow has the preparation problems of complex operation, low success rate, more related reagents, long period and the like. At present, no one-stop kit related to preparation and identification of mouse bone marrow macrophages is reported.
Disclosure of Invention
The invention aims to provide a mouse bone marrow macrophage preparation and detection kit, which can be used for mouse bone marrow macrophage separation culture and has the characteristics of integrating reagents required in experimental operation, being convenient to use, improving the yield of mouse bone marrow macrophages after use, ensuring stable cell adherence speed, low price of a detection method, low cost and the like;
the invention provides a kit for preparing mouse bone marrow macrophages, which comprises: the kit comprises a kit body, wherein a phosphate buffer solution (10 multiplied by PBS) reagent bottle, a streptomycin antibiotic reagent bottle, an erythrocyte lysate reagent bottle, a cell culture medium reagent bottle, a fetal bovine serum reagent bottle, a mouse fibroblast L929 culture supernatant reagent bottle, a CD11b antibody reagent bottle, a bacterial strain porcelain bead for phagocytosis identification and an instruction are filled in the kit body.
In the kit, the phosphate buffer solution reagent bottle contains a phosphate buffer solution reagent, and the phosphate buffer solution reagent consists of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and potassium chloride, wherein the sodium chloride is 1.37mol/L, the potassium chloride is 27mmol/L, the disodium hydrogen phosphate is 100mmol/L, the potassium dihydrogen phosphate is 18mmol/L, and the pH value is 7.4; it is diluted 10 times when used.
In the kit, the penicillin streptomycin antibiotic reagent bottle is filled with mixed antibiotic with the concentration of 10000U/ml penicillin G sodium and 10mg/ml streptomycin sulfate.
In the kit, the erythrocyte lysate reagent bottle is filled with the erythrocyte lysate reagent. The erythrocyte lysate reagent consists of 139.6mmol/L ammonium chloride, 16.96mmol/L tris (hydroxymethyl) aminomethane and pH 7.2.
In the kit, the cell culture medium reagent bottle contains the cell culture medium reagent, and the cell culture medium reagent contains a DMEM medium.
In the kit, the fetal bovine serum reagent bottle is filled with a fetal bovine serum reagent.
In the above kit, wherein the mouse fibroblast L929 culture supernatant reagent bottle contains a mouse fibroblast L929 culture supernatant reagent.
In the kit, the CD11c antibody reagent bottle contains fluorescein isothiocyanate labeled CD11b with the concentration of 0.5 mg/ml.
In the above kit, the phagocytic ability-identifying strain reagent bottle contains a phagocytic ability-identifying strain, and the phagocytic ability-identifying strain is escherichia coli having green fluorescence.
The invention also provides application of the kit in preparation of mouse bone marrow macrophages.
The invention overcomes the problems of time consuming, easy pollution, various reagent types and complex procedures in the current process of preparing and identifying the mouse bone marrow macrophages, and the preparation and identification kit for the mouse bone marrow macrophages integrates the reagents required in experimental operation, is convenient to use and optimizes the induction conditions of the mouse bone marrow cells. After the kit is used, the yield of mouse bone marrow macrophages can be improved, the cell adherence speed is stable, the detection method is low in price, the cost is low, and the like; the preparation efficiency and the survival rate of primary cell culture are greatly improved, the success rate of establishing a primary cell culture system is high, and preparation and identification are integrated to perform one-stop service.
Drawings
FIG. 1 is a schematic structural composition diagram of the kit of the present invention.
FIG. 2 is a diagram showing the state of cells cultured in vitro for 7 days using mouse bone marrow macrophages prepared according to the present invention.
FIG. 3 is a graph showing the identification of mouse bone marrow macrophage flow cytometer using the present invention
FIG. 4 is an immunofluorescence assay chart of a mouse bone marrow macrophage phagocytic "phagocytic ability assay strain" prepared by the present invention.
Detailed Description
The following examples are presented to enable those skilled in the art to more fully understand the present invention and are not intended to limit the invention in any way. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were all purchased from conventional biochemicals, unless otherwise specified.
Example 1: cell preparation and identification kit of the present invention
The embodiment of the tissue cell separation culture kit comprises a kit body 1, wherein a phosphate buffer solution (10 multiplied by PBS) reagent bottle 2, a streptomycin antibiotic reagent bottle 3, an erythrocyte lysate reagent bottle 4, a cell culture medium reagent bottle 5, a fetal bovine serum reagent bottle 6, a mouse fibroblast L929 culture supernatant reagent bottle 7, a fluorescein isothiocyanate labeled CD11b antibody reagent bottle 8, a phagocytosis capacity identification strain bottle 9 are filled in the kit body 1, and the kit further comprises an instruction 10.
The specific structure can be seen in FIG. 1, and FIG. 1 is a schematic structural composition diagram of the kit of the present invention.
The phosphate buffer solution (10 XPBS) reagent bottle is filled with a phosphate buffer solution (10 XPBS) reagent, the phosphate buffer solution reagent consists of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and potassium chloride, the sodium chloride is 1.37mol/L, the potassium chloride is 27mmol/L, the disodium hydrogen phosphate is 100mmol/L, the potassium dihydrogen phosphate is 18mmol/L, and the pH value is 7.4. When in use, the extract is diluted by 10 times;
the penicillin streptomycin antibiotic reagent bottle is filled with mixed antibiotic with the concentration of 10000U/ml penicillin G sodium and 10mg/ml streptomycin sulfate, and the mixed antibiotic is diluted by 100 times when in use;
in the kit, the erythrocyte lysate reagent bottle is filled with the erythrocyte lysate reagent. The erythrocyte lysate reagent consists of 139.6mmol/L ammonium chloride, 16.96mmol/L tris (hydroxymethyl) aminomethane and pH 7.2.
The cell culture medium reagent bottle is filled with a DMEM medium reagent.
The fetal calf serum reagent bottle is filled with a fetal calf serum reagent, and the volume ratio of a DMEM medium to the fetal calf serum is 9:1 when the fetal calf serum reagent bottle is used.
The mouse fibroblast L929 culture supernatant reagent bottle is filled with a mouse fibroblast L929 culture supernatant reagent, the mouse fibroblast L929 culture supernatant reagent plays a role in inducing mouse bone marrow cells to differentiate into macrophages, and is added into a cell culture medium when in use, and the volume ratio of a cell complete culture medium to the L929 culture supernatant is 4:1 when in use;
the fluorescein isothiocyanate labeled CD11b antibody reagent bottle is filled with a CD11b antibody with the concentration of 0.5mg/mL, CD11b is also called integrin, is expressed on the surfaces of monocytes and macrophages in a large quantity, and is diluted to 0.25 mu g/mL by phosphate buffer solution when used for immunofluorescence detection or flow cytometry staining detection when mouse bone marrow macrophage differentiation is successfully identified.
The bacterial strain reagent bottle for identifying the phagocytic ability is filled with bacterial strains for identifying the phagocytic ability, and the bacterial strains for identifying the phagocytic ability are Escherichia coli with green fluorescence. The host cell is BL21(DE3), the expression vector is pET30a, the target gene is gfp (GenBank: AAB08060.1), the experimental strain is named as BL21(DE3) (pET30a-gfp), and the experimental strain is cultured by a solid and liquid LB culture medium and induced by isopropyl thiogalactoside (IPTG) when in use;
the volume of the phosphate buffer solution (10 XPBS) reagent is 50ml, the label is attached, and the reagent is stored at 4 ℃.
The volume of the streptomycin antibiotic reagent is 1mL, and the reagent is labeled and stored at-20 ℃.
The volume of the erythrocyte lysate reagent is 20mL, and the erythrocyte lysate reagent is labeled and stored at room temperature.
The volume of the DMEM medium is 270mL, the DMEM medium is labeled, and the DMEM medium is stored at 4 ℃.
The volume of the fetal calf serum is 30mL, and the fetal calf serum is labeled and stored at-20 ℃.
60mL of culture supernatant of mouse fibroblast L929 used for inducing mouse bone marrow macrophages is labeled and stored at-20 ℃.
The volume of the fluorescein isothiocyanate labeled CD11b antibody reagent is 20 mu L, and the antibody reagent is sealed in a dark place, labeled and stored at the temperature of-20 ℃.
The bacterial strain for identifying the phagocytosis ability is stored as ceramic beads, and is labeled and stored at the temperature of minus 20 ℃.
The operation process comprises the following steps: sucking phosphate buffer solution from femur and tibia with injector, blowing out bone marrow, centrifuging, discarding supernatant, adding erythrocyte lysate, neutralizing with complete culture medium, centrifuging, discarding supernatant, blowing away cell precipitate with complete culture medium, spreading in disposable sterile bacteria culture dish, and placing in 5% CO at 37 deg.C2After 4 hours of culture in the incubator, the suspension cells were aspirated, centrifuged, suspended with complete medium of culture medium supplemented with mouse fibroblast L929 culture supernatant, placed in a cell culture dish, and placed at 37 ℃ in 5% CO2Culturing in an incubator for 7 days. The induced bacterial strain for identifying the phagocytosis ability can be added into the bone marrow macrophages after being cultured for 7 days for incubation, and then the phagocytosis ability is observed under a fluorescence microscope; the cell surface marker antigen can also be identified by flow cytometry after incubation with fluorescein isothiocyanate labeled CD11b antibody.
Example 2: mouse bone marrow macrophages prepared by using kit of the invention and identified by using the kit
The implementation takes the preparation and identification of mouse bone marrow macrophages as an example, and illustrates the application of the kit in the preparation and identification of mouse bone marrow macrophages. The kit used was the kit described in example 1.
The invention is further explained by the accompanying drawings and examples.
The preparation and identification steps are as follows:
1. dying a 7-week-old mouse by suffocation and euthanasia, disinfecting the surface of the mouse by using 75% ethanol, then operating on a sterile operating table with ultraviolet irradiation for 30 minutes, cutting an incision on retreating by using sterile surgical scissors, tearing skin to expose muscle, removing muscle tissues as much as possible by using the sterile scissors, and then taking a femur and a tibia;
2. shearing off two ends of femur and tibia with aseptic scissors to expose marrow cavity, washing marrow cavity with 2ml phosphate buffer solution containing 1% penicillin streptomycin antibiotic, repeating for several times until bone is white, blowing off marrow cells with a pipette, collecting washing solution, and placing into 15ml centrifuge tube; centrifuging at 800rpm for 5 minutes;
3. discarding the supernatant, re-suspending the cell precipitate with 2-3 mL of erythrocyte lysate, adding 10mL of complete culture medium for neutralization after 20s, centrifuging at 800RPM for 5min, and discarding the supernatant; (ii) a
4. Resuspending the cells, plating onto a bacterial culture dish, adding complete medium, and placing at 37 ℃ 5% CO2Culturing for 4 hours in an incubator;
5. the nonadherent suspension cells were aspirated, harvested by centrifugation, and added to complete medium containing 20% L929 cell culture supernatant and 1% streptomycin antibiotic at 1X 106Cell/ml Density cells were seeded in six well plates (34.8 mm pore size) and dishes were placed at 37 ℃ 5% CO2Culturing in an incubator;
6. every two days, cells were plated out using 2ml of complete medium containing 20% L929 cell culture supernatant per well;
7. at day 7, induced differentiation of mouse macrophages can be observed microscopically and photographed, see fig. 2.
8. At the 7 th day, identifying macrophage surface marker antigen of induced differentiated mouse macrophages, scraping cells in a six-hole plate by using a cell scraper, sucking cell suspension into a 15ml centrifuge tube, and centrifuging at 800rpm for 5 minutes; the cell concentration was adjusted to 1X 10 by counting7and/mL, adding 200. mu.L of the cell suspension into a 1.5mL centrifuge tube, adding 2. mu.L of fluorescein isothiocyanate-labeled CD11b antibody into the centrifuge tube, incubating the centrifuge tube for 30min at 4 ℃ in the dark, washing the centrifuge tube for 2 times with PBS, sieving the cells with a 300-mesh sieve, and identifying the cells by using a flow cytometer, and referring to FIG. 3.
9. At day 7, cells were characterized for their ability to phagocytose macrophages. Preparation items: drawing a bacterial strain for phagocytosis ability identification on an LB solid plate culture medium containing 30 mu g/mL kanamycin, and culturing for 12 hours in a constant-temperature incubator at 37 ℃; the next day, a single colony was picked up in 10mL of LB liquid medium containing 30. mu.g/mL kanamycin, cultured at 37 ℃ under 180rmp, and added to a final concentration of 1mM after 3 hoursIPTG induction is carried out, after 3 hours of induction, 1mL of bacterial liquid is sucked, centrifugation is carried out under the condition of 4000rmp for 5min, supernatant is removed, PBS is washed twice, and the concentration is set to be 1 x 10 by a turbidimetric method8And/ml. The cells were added to a mouse macrophage six-well plate at a ratio of multiplicity of infection of 10: 1. After 30min, the phagocytic capacity of mouse macrophages was observed under a fluorescent microscope. Viewing and collecting images under a fluorescence microscope see figure 4;
after the mouse bone marrow macrophages prepared by the method are cultured for 6 days, the cells are obviously attached to the wall and the cell morphology is consistent by observing the cells by a microscope (200X) as shown in figure 2. The cell morphology is consistent with that of macrophages, and the cell survival rate is high.
The method is used for preparing the mouse bone marrow macrophages, after the mouse bone marrow macrophages are cultured for 6 days, the mouse bone marrow macrophages are incubated for 30min by using fluorescein isothiocyanate to mark CD11b, 10000 cells are analyzed by a flow cytometer, the proportion of front scattered fluorescence (the size of a reaction cell) and side scattered fluorescence (the size and the number of particles contained in the reaction cell) is analyzed, and accordingly cell clusters are analyzed, the cultured cells belong to the same cluster, a small amount of aggregation and cell fragments are generated, and meanwhile, the positive rate of the CD11b on the cell surface of the mouse bone marrow macrophages is higher than 85%, and the reference of the figure 3 shows that the cultured cells belong to the same cluster.
After the mouse bone marrow macrophages prepared by the kit are cultured for 6 days, the bacterial strains for identifying the phagocytosis ability are used for carrying out phagocytosis experiments on the mouse bone marrow macrophages, and the phagocytosis experiments are observed and photographed under an immunofluorescence microscope (200 x), and a great amount of green fluorescence is found in cells, so that the mouse bone marrow macrophages prepared by the kit have the phagocytosis ability, and the phagocytosis experiments are shown in figure 4.
The invention overcomes the problems of time-consuming process, various reagent types, complex procedure and easy pollution in the existing preparation and identification of the mouse bone marrow macrophage. The mouse bone marrow macrophage preparation and identification kit is simple and convenient to operate, integrates most of reagents required in an experiment, optimizes induction conditions, greatly improves the preparation efficiency and the survival rate of primary cell culture, and has high success rate of establishing a primary cell culture system; the preparation and identification are integrated, so that a one-stop service is provided for the primary culture of the mouse macrophage, and the establishment of a primary culture system of the mouse macrophage is greatly facilitated.
Those skilled in the art will appreciate that the above embodiments are merely exemplary embodiments and that various changes, substitutions, and alterations can be made without departing from the spirit and scope of the invention.
Claims (10)
1. A kit for preparing mouse bone marrow macrophages, the kit comprising:
the kit comprises a kit body, wherein a phosphate buffer solution (10 multiplied by PBS) reagent bottle, a streptomycin antibiotic reagent bottle, an erythrocyte lysate reagent bottle, a cell culture medium reagent bottle, a fetal bovine serum reagent bottle, a mouse fibroblast L929 culture supernatant reagent bottle, a CD11b antibody reagent bottle, a Tween 20 reagent bottle, a phagocytosis capacity identification strain bottle and an instruction book are filled in the kit body.
2. The kit of claim 1, wherein the phosphate buffer reagent bottle contains a phosphate buffer reagent consisting of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, and potassium chloride, sodium chloride 1.37mol/L, potassium chloride 27mmol/L, disodium hydrogen phosphate 100mmol/L, potassium dihydrogen phosphate 18mmol/L, pH 7.4.
3. The kit of claim 1, wherein the penicillin streptomycin antibiotic reagent bottle contains a mixed antibiotic with a concentration of 10000U/ml penicillin G sodium and 10mg/ml streptomycin sulfate.
4. The kit of claim 1, wherein the red blood cell lysate reagent bottle contains a red blood cell lysate reagent consisting of 139.6mmol/L ammonium chloride, 16.96mmol/L tris, pH 7.2.
5. The kit of claim 1, wherein the cell culture media reagent bottles contain cell culture media reagent containing DMEM cell culture media.
6. The kit of claim 1, wherein the fetal bovine serum reagent bottle contains fetal bovine serum reagent.
7. The kit according to claim 1, wherein the mouse fibroblast L929 culture supernatant reagent bottle contains mouse fibroblast L929 culture supernatant reagent.
8. The kit of claim 1, wherein the CD11b antibody reagent vial contains fluorescein isothiocyanate labeled CD11b antibody at a concentration of 0.5 mg/ml.
9. The kit according to claim 1, wherein the phagocytic ability-identifying strain reagent bottle contains a phagocytic ability-identifying strain, and the phagocytic ability-identifying strain is Escherichia coli having green fluorescence.
10. Use of a kit according to any one of claims 1 to 9 in the preparation of mouse bone marrow macrophages.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114891718A (en) * | 2022-06-08 | 2022-08-12 | 天康制药(苏州)有限公司 | Culture medium for suspension culture of bone marrow cells, preparation method, application and method for inducing differentiation of bone marrow-derived cells into macrophages |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100003272A1 (en) * | 2007-01-11 | 2010-01-07 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Method for Expanding Monocytes |
| CN103977029A (en) * | 2014-04-11 | 2014-08-13 | 中国人民解放军第四军医大学 | Application of classically activated macrophages in treatment of liver fibrosis |
| CN110447601A (en) * | 2019-08-09 | 2019-11-15 | 南京医科大学 | A kind of targeting macrophage is overexpressed the preparation method of genetic mouse model |
| CN110669731A (en) * | 2019-10-22 | 2020-01-10 | 南通大学 | Method for separating inherent and infiltrated macrophages of liver |
-
2020
- 2020-02-13 CN CN202010090033.5A patent/CN111254112A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100003272A1 (en) * | 2007-01-11 | 2010-01-07 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Method for Expanding Monocytes |
| CN103977029A (en) * | 2014-04-11 | 2014-08-13 | 中国人民解放军第四军医大学 | Application of classically activated macrophages in treatment of liver fibrosis |
| CN110447601A (en) * | 2019-08-09 | 2019-11-15 | 南京医科大学 | A kind of targeting macrophage is overexpressed the preparation method of genetic mouse model |
| CN110669731A (en) * | 2019-10-22 | 2020-01-10 | 南通大学 | Method for separating inherent and infiltrated macrophages of liver |
Non-Patent Citations (5)
| Title |
|---|
| FERNANDA M. MARIM: "A Method for Generation of Bone Marrow-Derived Macrophages from Cryopreserved Mouse Bone Marrow Cells", 《PLOS ONE》 * |
| JOACHIM WEISCHENFELDT ET AL.: "Bone Marrow-Derived Macrophages (BMM): Isolation and Applications", 《COLD SPRING HARB. PROTOC.》 * |
| VIRGINIE TROUPLIN ET AL.: "Bone Marrow-derived Macrophage Production", 《JOURNAL OF VISUALIZED EXPERIMENTS》 * |
| 习大林等: "BLP耐受通过上调MARCO增强巨噬细胞的吞噬能力", 《中国病理生理杂志》 * |
| 林静等: "Maresin-1通过ALX/NF-κB通路促进巨噬细胞的自噬", 《温州医科大学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114891718A (en) * | 2022-06-08 | 2022-08-12 | 天康制药(苏州)有限公司 | Culture medium for suspension culture of bone marrow cells, preparation method, application and method for inducing differentiation of bone marrow-derived cells into macrophages |
| CN114891718B (en) * | 2022-06-08 | 2024-01-30 | 天康制药股份有限公司 | Culture medium for suspension culture of bone marrow cells, preparation method, application and method for inducing differentiation of bone marrow-derived cells into macrophages |
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