CN110713969A - Blue crab blood cell grouping culture method - Google Patents
Blue crab blood cell grouping culture method Download PDFInfo
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- CN110713969A CN110713969A CN201910978424.8A CN201910978424A CN110713969A CN 110713969 A CN110713969 A CN 110713969A CN 201910978424 A CN201910978424 A CN 201910978424A CN 110713969 A CN110713969 A CN 110713969A
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Abstract
The invention discloses a method for culturing blue crab blood cell groups, which comprises the following steps: sterilizing the surfaces of the blue crabs; sucking blue crab blood, and mixing with the anticoagulant; adding the blood cell suspension into a centrifuge tube filled with the cell separation liquid, and performing density gradient centrifugation to obtain layering liquid of three cell subsets; the preparation method of the cell separation solution comprises the following steps: sequentially adding 80%, 60% and 30% of Percoll cell separation solution with the same volume in a centrifuge tube; respectively sucking cell suspensions of three different cell subsets, placing in different cell culture solutions, and culturing at 28 deg.C without CO2Primary cell culture was performed under the conditions of addition. The method adopts specific anticoagulant and improved cell separating medium and cell culture medium, has simple operation, strong repeatability and stable cell growth, and obviously improves the separation effect of three different cell subsets.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a method for blue crab blood cell grouping culture.
Background
With the rapid development of life science and technology, cell engineering has been increasingly emphasized. Culturing primary cells as a research material has been an important aspect in the scientific fields of cell biology, molecular biology, toxicology, and the like. The crustacean has no mature cell line at present because the research related to cell culture starts late, so that the establishment of a stable primary cell culture system is a hotspot of the research on the cell culture of the crustacean at present.
The crustacean blood cells are important immune cells, participate in cellular immunity and humoral immunity, including phagocytosis, encapsulation, blackening and bacteriolysis, and release of cell factors such as antibacterial peptide, lectin, phenol oxidase and cell adhesion protein, and are important components of crustacean innate immunity. Crustacean blood cells are composed of a variety of cells, and are generally classified into Hyalinocyte (HC), hemi-granule (SGC) and Granule (GC) cells, depending on the number of granules in the cytoplasm and the nucleoplasm ratio. Different cell types have different functions, the clear cells mainly have the functions of identifying pathogen and phagocytosis, the half-granule cells have the functions of encapsulation, storing and releasing prophenoloxidase, and the granule cells also have the functions of storing and releasing prophenoloxidase and have cytotoxicity.
The blue crabs are important economic seawater varieties in coastal areas of China, but serious disease threats are brought by overhigh breeding density, so that the morbidity and mortality of the blue crabs are high, and effective disease control approaches and effective medicines are urgently needed. The establishment of the blue crab blood cell grouping culture method can lay a foundation for the research in the fields of crustacean cytobiology, immunology and the like, and can also provide an in vitro cell model for drug screening.
Disclosure of Invention
The invention provides a method for culturing blue crab blood cell groups, which is simple to operate, strong in repeatability and stable in cell growth, can separate three different cell subgroups more efficiently, and lays a foundation for research in the fields of crustacean cytobiology, immunology and the like.
The specific technical scheme is as follows:
a method for blue crab blood cell grouping culture comprises the following steps:
(1) preparing an anticoagulant, a cell separation solution and a cell culture solution of the blue crab blood;
(2) cleaning the blue crabs with seawater, and then disinfecting the surfaces of the blue crabs;
(3) sucking blue crab blood, and mixing the blue crab blood with the anticoagulant to obtain blood cell suspension; adding the blood cell suspension into a centrifuge tube filled with the cell separation liquid, and performing density gradient centrifugation to obtain three layered liquids of cell subsets, namely transparent cells, half-granular cells and granular cells from top to bottom;
the formula of the anticoagulant comprises: 4.5-5.0g/L citric acid, 13.0-13.5g/L sodium citrate, 14.5-15.0g/L glucose and 1.0-1.5g/L sodium chloride;
the preparation method of the cell separation solution comprises the following steps: sequentially adding 80%, 60% and 30% of Percoll cell separation solution with the same volume in a centrifuge tube;
(4) respectively sucking cell suspensions of three different cell subsets, placing in different cell culture solutions, and culturing at 28 deg.C without CO2Performing primary cell culture under the condition of addition;
the formula of the cell culture solution is as follows: taking L15 culture medium as basic culture medium, adding 0.2mM sodium chloride, 100U/mL penicillin, 100 μ g/mL streptomycin and 5-10% volume fraction fetal calf serum, pH 7.4-7.6.
The invention adopts specific anticoagulant and improved cell separating medium and cell culture medium, not only has simple operation, strong repeatability and stable cell growth, but also obviously improves the separation effect of three different cell subsets.
Preferably, in the step (2), the seawater is sterilized, and the salinity is 25-35 ppt.
Preferably, in the step (2), the surfaces of the blue crabs are disinfected by using an ethanol water solution with the volume fraction of 70-80%.
Preferably, in the step (3), the volume ratio of the blue crab blood to the anticoagulant is 1: 1.
Preferably, in the step (3), the density gradient centrifugation is carried out under 1200-1500 g centrifugation for 25-35 minutes.
Preferably, in step (4), half the volume of the cell culture solution is replaced every two days, and the cell culture solution is moved to an inverted phase-contrast microscope every 6 hours to observe the state of the cells.
Preferably, the blue crab is Scylla paramamosain.
Compared with the prior art, the invention has the following beneficial effects:
(1) the method adopts specific anticoagulant and improved cell separating medium and cell culture medium, has simple operation, strong repeatability and stable cell growth, and obviously improves the separation effect of three different cell subsets.
(2) The invention establishes a stable primary culture system of the blue crab blood cells, and lays a foundation for the research in the fields of crustacean cell biology, immunology and the like.
(3) The invention is beneficial to researching the innate immunity of the blue crabs under the in vitro condition, and provides help for the disease control way and the drug development of the blue crabs.
Drawings
FIG. 1 is a photograph during the separation of different cell subsets in example 1;
wherein A is a layering result after density gradient centrifugation, and cell Debris (Debris), clear cells (HC), half granular cells (SGC) and Granular Cells (GC) are respectively arranged from top to bottom; hemolymph represents hemolymph; percoll is the name of the cell separation fluid;
b is a graph of the results of identifying different cell subsets under a fluorescent microscope after staining with DAPI.
FIG. 2 is a photomicrograph of a culture of primary cells of different cell subsets of example 1;
wherein A is the result of culturing the hyaline cells for 24 hours; b is the result of half-granular cell culture after 24 hours; c is the result of the granular cells after 24h of culture.
Detailed Description
The present invention will be further described with reference to the following specific examples, which are only illustrative of the present invention, but the scope of the present invention is not limited thereto.
Example 1
A scylla paramamosain blood cell grouping culture method comprises the following specific steps:
(1) preparing an anticoagulant, a cell separation solution and a cell culture solution of Scylla paramamosain blood;
(2) after the green mud crabs are cleaned by seawater with sterile salinity of 30ppt, the surfaces of the green mud crabs are disinfected by an ethanol water solution with volume fraction of 75 percent;
(3) sucking the scylla paramamosain blood by using a syringe, and mixing the scylla paramamosain blood with the anticoagulant to obtain a blood cell suspension; adding the blood cell suspension into a centrifuge tube filled with the cell separation liquid, performing density gradient centrifugation, and centrifuging 1400g for 30 minutes to obtain three cell subsets of layered liquids, namely transparent cells, half-granular cells and granular cells from top to bottom;
the formula of the anticoagulant comprises: 4.8g/L citric acid, 13.2g/L sodium citrate, 14.7g/L glucose and 1.2g/L sodium chloride; mixing the scylla paramamosain blood and the anticoagulant in a volume ratio of 1: 1;
the preparation method of the cell separation solution comprises the following steps: sequentially adding 80%, 60% and 30% of Percoll cell separation solution with the same volume in a centrifuge tube;
(4) respectively sucking cell suspensions of three different cell subsets, placing in different cell culture solutions, and culturing at 28 deg.C without CO2Performing primary cell culture under the condition of addition;
the formula of the cell culture solution is as follows: taking an L15 culture medium as a basic culture medium, adding 0.2mM sodium chloride, 100U/mL penicillin, 100 mu g/mL streptomycin and 5-10% by volume of fetal calf serum, and adjusting the pH to 7.4-7.6; half of the volume of the cell culture solution was changed every two days, and the cell culture solution was moved to an inverted phase contrast microscope every 6 hours to observe the cell state.
The results are shown in FIGS. 1 and 2.
Example 2
In the test process, the influence of separation liquids under different conditions on the separation effect is researched; the conditions were the same as in example 1 except that density gradient centrifugation was performed using different separation solutions, and the results are shown in Table 1.
TABLE 1
Claims (7)
1. A method for blue crab blood cell grouping culture is characterized by comprising the following steps:
(1) preparing an anticoagulant, a cell separation solution and a cell culture solution of the blue crab blood;
(2) cleaning the blue crabs with seawater, and then disinfecting the surfaces of the blue crabs;
(3) sucking blue crab blood, and mixing the blue crab blood with the anticoagulant to obtain blood cell suspension; adding the blood cell suspension into a centrifuge tube filled with the cell separation liquid, and performing density gradient centrifugation to obtain three layered liquids of cell subsets, namely transparent cells, half-granular cells and granular cells from top to bottom;
the formula of the anticoagulant comprises: 4.5-5.0g/L citric acid, 13.0-13.5g/L sodium citrate, 14.5-15.0g/L glucose and 1.0-1.5g/L sodium chloride;
the preparation method of the cell separation solution comprises the following steps: sequentially adding 80%, 60% and 30% of Percoll cell separation solution with the same volume in a centrifuge tube;
(4) respectively sucking cell suspensions of three different cell subsets, placing in different cell culture solutions, and culturing at 28 deg.C without CO2Performing primary cell culture under the condition of addition;
the formula of the cell culture solution is as follows: taking L15 culture medium as basic culture medium, adding 0.2mM sodium chloride, 100U/mL penicillin, 100 μ g/mL streptomycin and 5-10% volume fraction fetal calf serum, pH 7.4-7.6.
2. The method for blue crab blood cell mass culture according to claim 1, wherein in the step (2), the seawater is sterilized and has a salinity of 25-35 ppt.
3. The method for culturing blue crab blood cell groups according to claim 1, wherein in the step (2), the surface of the blue crab is disinfected by using an ethanol aqueous solution with a volume fraction of 70-80%.
4. The method for blue crab blood cell population culture according to claim 1, wherein in the step (3), the blue crab blood is mixed with the anticoagulant at a volume ratio of 1: 1.
5. The method for culturing blue crab blood cell clusters according to claim 1, wherein in the step (3), the density gradient centrifugation is carried out under 1200-1500 g centrifugation for 25-35 minutes.
6. The method for culturing blue crab blood cell mass according to claim 1, wherein in the step (4), half of the volume of the cell culture solution is changed every two days, and the cell culture solution is moved to an inverted phase contrast microscope to observe the cell state every 6 hours.
7. The method of claim 1, wherein the blue crab is Scylla Paramamosain.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114134099A (en) * | 2021-11-29 | 2022-03-04 | 北部湾大学 | Balanced salt solution of blood cells of marine invertebrates |
| CN115074306A (en) * | 2022-07-12 | 2022-09-20 | 中国海洋大学 | Culture medium for long-term culture and continuous passage of crab cells |
Citations (2)
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| CN103288955A (en) * | 2013-05-16 | 2013-09-11 | 青岛农业大学 | Monoclonal antibody of anti-blue crab particle hemocyte 26.7kDa protein, and preparation method thereof |
| CN108118024A (en) * | 2017-11-22 | 2018-06-05 | 宁波大学 | A kind of Portunus trituberculatus Miers haemocyte separation method |
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2019
- 2019-10-15 CN CN201910978424.8A patent/CN110713969A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103288955A (en) * | 2013-05-16 | 2013-09-11 | 青岛农业大学 | Monoclonal antibody of anti-blue crab particle hemocyte 26.7kDa protein, and preparation method thereof |
| CN108118024A (en) * | 2017-11-22 | 2018-06-05 | 宁波大学 | A kind of Portunus trituberculatus Miers haemocyte separation method |
Non-Patent Citations (3)
| Title |
|---|
| YI-LIANZHOU: "Hemocytes of the mud crab Scylla paramamosain: Cytometric, morphological characterization and involvement in immune responses", 《FISH & SHELLFISH IMMUNOLOGY》 * |
| YI-LIANZHOU: "Identification and functional analysis of immune deficiency (IMD) from Scylla paramamosain: The first evidence of IMD signaling pathway involved in immune defense against bacterial infection in crab species", 《FISH & SHELLFISH IMMUNOLOGY》 * |
| 乔琨: "拟穴青蟹血细胞原代培养及条件优化", 《中国畜牧兽医》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114134099A (en) * | 2021-11-29 | 2022-03-04 | 北部湾大学 | Balanced salt solution of blood cells of marine invertebrates |
| CN114134099B (en) * | 2021-11-29 | 2023-06-16 | 北部湾大学 | Balanced salt solution for blood cells of marine invertebrate |
| CN115074306A (en) * | 2022-07-12 | 2022-09-20 | 中国海洋大学 | Culture medium for long-term culture and continuous passage of crab cells |
| CN115074306B (en) * | 2022-07-12 | 2023-09-22 | 中国海洋大学 | Culture medium for long-term culture and continuous passage of crab cells |
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