CN114958742B - Method for separating spleen macrophages of grass carp - Google Patents
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Abstract
The invention provides a method for separating grass carp spleen macrophages, which comprises the following steps: taking out spleen tissues of grass carp, and cleaning the spleen tissues; immersing the cleaned spleen tissue in a sample diluent and grinding the spleen tissue to obtain a tissue homogenate suspension; adding the tissue homogenate suspension into digestive juice, and performing shake digestion after adding to obtain a digestion product; filtering and centrifuging the digested product to obtain a first product, and culturing the first product after counting and counting the plates to obtain spleen macrophages of the grass carp. The fish spleen macrophages separated by the method have the advantages of large number, good cell state and high success rate.
Description
Technical Field
The invention relates to the technical field of cell separation, in particular to a method for separating spleen macrophages of grass carp.
Background
Primary cell culture is an important means of currently studying specific physiological functions of fish immune cells. However, the culture operation of the lower vertebrate cells is complicated, the cell yield is low, the cost is high, and the separation of the spleen macrophages of the fishes is not disclosed in the published patent at present. If the fish cell culture is performed by referring to the primary macrophage culture of the spleen of the mammal, the spleen of the mammal is smaller than that of the mammal, so that the yield of the spleen macrophages is too low (the total number of the isolated white blood cells is generally 1-5×10≡6, and the wall of a general western blotting experiment needs about 1×10≡7 leukocytes/well to obtain enough macrophages), therefore, the traditional separation method of combining physical grinding with the lymph separation liquid of the mammal cannot be fully suitable for the fishes. Therefore, development of an efficient and stable fish spleen primary macrophage separation method is urgent.
Disclosure of Invention
The invention aims to provide a method for separating spleen macrophages of grass carp, so as to solve the problems.
In order to achieve the above purpose, the embodiment of the present application provides the following technical solutions:
a method of isolating grass carp spleen macrophages, comprising:
taking out spleen tissues of grass carp, and cleaning the spleen tissues;
immersing the cleaned spleen tissue in a sample diluent and grinding the spleen tissue to obtain tissue homogenate;
adding the tissue homogenate into digestive juice, and performing shake digestion after adding to obtain a digestion product;
filtering and centrifuging the digested product to obtain a first product, and culturing the first product after counting and counting the plates to obtain spleen macrophages of the grass carp.
Optionally, adding the tissue homogenate to the digestive juice comprises:
adding the tissue homogenate into a first container, adding a sample diluent or a culture medium into the first container until the total volume is a first value, adding collagenase, dispase and triple antibody into the first container after adding the sample diluent or the culture medium, wherein the adding amount of collagenase, dispase and triple antibody is equal.
Alternatively, the first value is 9.7mL, and the collagenase, dispase and tri-antibody are added in amounts of 100uL.
Optionally, the adding is followed by shake digestion, including:
digestion was carried out after addition in a water bath shaker at a water bath temperature of 30 ℃.
Optionally, the digestion is performed in a water bath shaker at a water bath temperature of 30 ℃ after the addition, comprising:
after addition, the mixture was digested for 40-50min in a water bath shaker at 30℃and at a shaking speed of 100rpm/min.
Optionally, filtering and centrifuging the digested product to obtain a first product, including:
the digested product is passed through a first screen to obtain a first filtered material, the first filtered material is transferred into a second container after being resuspended and is supplemented to 20mL by a sample diluent, and the first filtered material and the second filtered material are uniformly mixed to obtain a first mixture;
adding the first mixture into two third containers on average, pre-filling 10mL of lymphocyte separation liquid in each third container, balancing after adding, centrifuging for the first time after balancing, and taking out substances in critical layers of the lymphocyte separation liquid and the diluent after centrifuging to obtain a second mixture;
and adding the second mixture into a fourth container pre-filled with sample diluent, balancing after adding, centrifuging for the second time after balancing, removing the supernatant in the fourth container after centrifuging, and re-suspending after removing to obtain a first product.
Optionally, in the first centrifugation process, using a basket centrifuge to centrifuge for 15 minutes at a temperature of 22 ℃ and a rotation speed of 2950 rpm; during the second centrifugation, the basket centrifuge was used for centrifugation at a temperature of 22℃and a rotational speed of 2390rpm for 2 minutes.
Optionally, culturing the first product after counting and counting the plates to obtain spleen macrophages of the grass carp, including:
counting the first product and performing plate spotting operation, placing the first product into a cell incubator for culturing to obtain macrophages, and performing cell cleaning treatment after culturing to obtain adherent macrophages;
observing whether the adherent macrophages meet the requirement of continuous culture, and if so, continuously culturing by using a culture medium to obtain the spleen macrophages of the grass carp.
Alternatively, the medium is RPMI1640 medium containing 10% fbs and 1% tri-antibody.
Optionally, if the result is met, the culture medium is used for further culturing for at least 2 hours, and the spleen macrophages of the grass carp are obtained.
The beneficial effects of the invention are as follows:
1. the fish spleen is coated with thin connective tissue, and collagenase can specifically hydrolyze the three-dimensional helical structure of collagen in connective tissue under physiological pH and temperature conditions without damaging other proteins and tissues, so the invention uses collagenase-II to digest collagen in the spleen, thereby facilitating the separation of various cells in solid tissues; dispase-II can be used to prepare cells from a variety of different tissues and organs. dispase-II has proven to be a fast, efficient and gentle reagent useful for isolating many tissues and cells grown in vitro, and is particularly useful for tissue dissociation and subculture since it does not disrupt the cell membrane. Therefore, compared with the traditional mechanical method, the method can obviously improve the yield of spleen macrophage white blood cells.
2. The fish spleen macrophages separated by the method have the advantages of large number, good cell state and high success rate.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the embodiments of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims thereof as well as the appended drawings.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate and do not limit the invention.
FIG. 1 is a photograph of a grass carp spleen cell suspension isolated by mechanical means;
FIG. 2 is a photograph of a spleen cell suspension of grass carp obtained by the separation method in example 1;
FIG. 3 is a photograph of cells when culture has not been started after completion of plating;
FIG. 4 is a photograph of adherent macrophages after 3h of culture;
FIG. 5 is a photograph of spleen macrophages of grass carp obtained after culturing the adherent macrophages for 48 hours;
FIG. 6 is a photograph showing morphological identification of spleen macrophages of grass carp obtained after culturing the adherent macrophages for 48 hours.
Detailed Description
All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The experimental methods used in the embodiments of the present invention are all conventional methods unless specifically described.
The invention provides a method for separating grass carp spleen macrophages, which comprises the following steps:
(one) animal implementation: healthy grass carp around 700g
(II) experimental reagent: rat lymph isolates (Tianjin, ocean), RPMI1640 medium (Gibco), inactivated fetal bovine serum FBS (Gibco), collagenase II (sigma),disperse enzyme II (sigma), tri-antibodies (penicillin, streptomycin and dihydromycin, aladin), PBS (Gibco);
and (III) implementing the steps:
3.1 preparation:
(1) 2, sterilizing surgical scissors (one each with the diameter of 8cm and the size of 16 cm), 1 forceps, 1 cell screen and 1 cell separation pestle in advance (121 ℃ C., 30 min);
(2) Preparing a rat lymph separating liquid, inactivating test reagents and consumables such as PBS, RPMI1640 culture medium, BD tube, cell screen (200 meshes), 60 culture dishes, cell culture plates, pasteur pipettes and the like;
3.2 taking spleen: after the grass carp is anesthetized, the spine is quickly broken, the whole viscera of the grass carp are dissected, the whole spleen is carefully taken out along the connective tissue of the spleen and the liver pancreas (note that the normal spleen is bright red (the spleen of the diseased fish is red and black), the edges and corners of the organs are clear, the texture is compact and rich in elasticity, and the grass carp is not in a swelling erosion state), and the grass carp is placed in a culture dish with a sample diluent or RPMI1640 culture medium, and the spleen tissue is completely immersed by tilting and light shaking.
3.3 discarding the culture medium, adding 3mL of RPM 1640 culture medium to wash the spleen, discarding the culture medium, adding 1-2mL of sample diluent, and shearing spleen tissues by using small surgical scissors; the spleen tissue is extruded by a cell separation pestle to achieve tissue homogenate (uniform paste state, no granular sense), so that the subsequent digestion is convenient, the tissue is extruded vertically as much as possible, the pestle rod is not pushed to avoid mechanical damage to cells, cell fragments are generated, and the cell yield is reduced; pressing spleen tissue requires repeated times.
3.4 digestion of spleen tissue
Transferring spleen tissue homogenate into a first container (50 mL BD tube), adding a proper amount of sample diluent (or RPMI1640 culture medium) until the total volume is 9.7mL, adding 100uL collagenase II, 100uL dispersing enzyme II and 100uL three-antibody, and digesting for 40-50min in a water bath shaking table (100 rpm/min) at 30 ℃ after adding, wherein the step obviously improves the white blood cell yield of spleen macrophages compared with the traditional mechanical method. The fish spleen is coated with thin connective tissue, and collagenase can specifically hydrolyze the three-dimensional helical structure of collagen in connective tissue under physiological pH and temperature conditions without damaging other proteins and tissues, so that the collagen in the spleen is digested by collagenase-II, and various cells in solid tissues are easy to separate; dispase-II has proven to be a fast, efficient and gentle reagent that can be used to isolate many tissues and cells grown in vitro; the dispase is particularly suitable for tissue dissociation and subculture because it does not damage cell membranes.
3.5, the digestion product obtained after the digestion in the previous step, namely, the spleen cell suspension of the grass carp is filtered by a 200-mesh screen, the filtered spleen cell suspension of the grass carp, namely, the first filter material is transferred into a second container, namely, a new BD tube, and is supplemented to 20mL by a sample diluent, and the mixture is fully and uniformly mixed to form a cell suspension, namely, a first mixture; 10mL of the cell suspension was slowly added along the tube wall with a 1mL pipette to two BD tubes each pre-filled with 10mL of lymphocyte separation medium, the third vessel, respectively, and this step was noted: the cell suspension is slowly added to the surface of the lymphocyte separation medium along the tube wall, and the cell suspension is arranged on the upper layer of the lymphocyte separation medium and cannot damage the interface between the cell suspension and the lymphocyte separation medium when being added later.
3.6 two BD tubes with lymphocyte isolates and cell suspensions added were removed, and after trimming, basket centrifuges were centrifuged at 2950rpm for 15 minutes at 22℃in acel: 6, brake: 4).
3.7 taking BD tube, at this time, spleen mixed white blood cells were near white flocculent cell layer (critical layer of lymphocyte separation solution and diluent) on top of lymphocyte separation solution, gently sucking out the layer of cells, i.e. second mixture, by Pasteur pipette in fourth vessel BD tube with sample diluent added, and centrifuging at 2390rpm at 22℃for 2 minutes.
3.8 removing supernatant, adding 5mL RPMI1640 medium (containing 1% triple antibody and 1% FBS) to resuspend cells, get the first product, taking 5uL of the first product for cell counting.
3.9 dot plate: taking a 6well plate as an example, cells in each well generally require 2X 10≡7 spleen white blood cells (the cell density required for other well plate cultures can be calculated therefrom), 28℃cell incubator (5% CO 2 ) Culturing for 2-3h。
Observing the adherence condition under a microscope, after adherence, washing off unadhered cells, washing for 2-3 times by using PBS, observing the occupation ratio of the adherent macrophages and the morphology of the cells under the microscope, if the occupation ratio of the adherent macrophages to each well is 50-70% and the morphology of the cells is normal (normal grass carp spleen macrophages are nearly irregular round or oval, the edges of the cells are always protruded, the cells are in a normal state when no vacuoles or less vacuoles exist, the cells are abnormal when the vacuoles are serious), continuously culturing by using an RPMI1640 culture medium containing 10% FBS and 1% triple antibody for at least 2 hours, and finally obtaining the grass carp spleen macrophages.
The invention has been tested several times in succession, and the invention will now be described in further detail with reference to a few test results, which are described in detail below in connection with specific examples.
Example 1
The embodiment provides a method for separating spleen macrophages of grass carp, which comprises the following steps:
(one) animal implementation: 703g of healthy grass carp
(II) experimental reagent: rat lymph isolates (Tianjin, ocean), RPMI1640 medium (Gibco), inactivated fetal bovine serum FBS (Gibco), collagenase II (sigma),disperse enzyme II (sigma), tri-antibodies (penicillin, streptomycin and dihydromycin, aladin), PBS (Gibco);
and (III) implementing the steps:
3.1 preparation:
(1) 2, sterilizing surgical scissors (one each with the diameter of 8cm and the size of 16 cm), 1 forceps, 1 cell screen and 1 cell separation pestle in advance (121 ℃ C., 30 min);
(2) Preparing a rat lymph separating liquid, inactivating test reagents and consumables such as PBS, RPMI1640 culture medium, BD tube, cell screen (200 meshes), 60 culture dishes, cell culture plates, pasteur pipettes and the like;
3.2 taking spleen: after the grass carp is anesthetized, the spine is rapidly broken, the whole viscera of the grass carp are dissected, the whole spleen is carefully taken out along the connective tissue of the spleen and the liver pancreas, and the whole spleen is placed in a culture dish with a sample diluent or RPMI1640 culture medium, and the whole spleen tissue is completely immersed by tilting and shaking the disc slightly.
3.3 discarding the culture medium, adding 3mL of RPM 1640 culture medium to wash the spleen, discarding the culture medium, adding 1-2mL of sample diluent, and shearing spleen tissues by using small surgical scissors; the spleen tissue is extruded by a cell separation pestle to achieve tissue homogenate (uniform paste state, no granular sense), so that the subsequent digestion is convenient, the tissue is extruded vertically as much as possible, the pestle rod is not pushed to avoid mechanical damage to cells, cell fragments are generated, and the cell yield is reduced; pressing spleen tissue requires repeated times.
3.4 digestion of spleen tissue
Transferring spleen tissue homogenate into a first container (50 mL BD tube), adding a proper amount of sample diluent (or RPMI1640 culture medium) to a total volume of 9.7mL, adding 100uL of collagenase II, 100uL of dispersing enzyme II and 100uL of triple antibody, adding, and then digesting for 50min in a water bath shaker (100 rpm/min) at 30 ℃ to obtain grass carp spleen cell suspension after digestion, wherein as shown in figure 2, figure 1 is the grass carp spleen cell suspension obtained by adopting a mechanical separation method. By observing FIGS. 1 and 2 (the illustrations in FIGS. 1 and 2 are abbreviated as: E: erythrocytes; L: leukocytes), it is evident that this step significantly improves spleen macrophage leukocyte yield over conventional mechanical methods.
3.5 passing the digested product obtained by the digestion in the above step, namely, the spleen cell suspension of the grass carp through a 200-mesh cell screen, transferring the filtered spleen cell suspension of the grass carp, namely, the first filtrate, into a second container, namely, a new BD tube, supplementing 20mL with sample diluent, fully and uniformly mixing to form a cell suspension, namely, a first mixture, slowly adding 10mL of cell suspension into two BD tubes, namely, a third container, which are respectively filled with 10mL of lymphocyte separation liquid along the tube wall by using a 1mL pipetting gun, wherein the following steps are as follows: the cell suspension is slowly added to the surface of the lymphocyte separation medium along the tube wall, and the cell suspension is arranged on the upper layer of the lymphocyte separation medium and cannot damage the interface between the cell suspension and the lymphocyte separation medium when being added later.
3.6 two BD tubes with lymphocyte isolates and cell suspensions were centrifuged at 2950rpm for 15 min at 22℃C (acel: 6, brake: 4).
3.7 removing BD tube, at this time, spleen mixed white blood cells were near white flocculent cell layer (critical layer of lymphocyte separation solution and dilution solution) on top of lymphocyte separation solution, and this layer of cells, second mixture, was gently sucked out by Pasteur pipette into BD tube of fourth container to which sample dilution solution had been added, and centrifuged at 2390rpm at 22℃for 2 minutes.
3.8 supernatant was discarded, 5mL of RPMI1640 medium (containing 1% tri-antibody and 1% FBS) was used to resuspend the cells to give a first product, and 5uL of the first product was used for cell counting.
3.9 dot plate: a photograph of cells from a 6well plate, in which 1.24X10-8 spleen leukocytes were implanted in each well, was taken after the end of the plating and had not been cultured, and is shown in FIG. 3. The cells were cultured in a cell incubator at 28℃for 3 hours.
3.10 culturing in a cell incubator for 3h, observing the adherence condition under a microscope, washing off non-adherence cells after adherence, washing with PBS for 2 times, and observing the occupation ratio of the adherence macrophages and the morphology of the cells under the microscope, wherein the occupation ratio of the adherence macrophages in the embodiment is 50-70% of the area of each well and the morphology of the cells is normal as shown in fig. 4;
3.11 the adherent macrophages obtained above were further cultured for 48 hours using RPMI1640 medium containing 10% fbs, 1% tri-antibodies (penicillin, streptomycin and dihydromycin), and finally spleen macrophages of grass carp were obtained. After 48 hours of culture, the cells were subjected to morphological identification by Giemsa starting as shown in FIG. 5, and the identification result was shown in FIG. 6. It can be seen that the cell morphology and size are in accordance with the characteristics of fish macrophages: the nucleus is mauve, the cytoplasm is bluish, and a small amount of vacuoles are occasionally formed; the nucleus is deviated and kidney-shaped, and the nuclear diameter accounts for about 1/3-1/2 of the cell.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A method of isolating grass carp spleen macrophages, comprising:
taking out spleen tissues of grass carp, and cleaning the spleen tissues;
immersing the cleaned spleen tissue in a sample diluent and grinding the spleen tissue to obtain tissue homogenate;
adding the tissue homogenate into digestive juice, and performing shake digestion after adding to obtain digestion products, wherein the shake digestion after adding comprises digestion in a water bath shaker at a water bath temperature of 30 ℃;
filtering and centrifuging the digested product to obtain a filtered and centrifuged product, and culturing the filtered and centrifuged product after counting and counting the plates to obtain spleen macrophages of the grass carp;
wherein adding the tissue homogenate to the digestive juice comprises:
adding the tissue homogenate into a BD tube, adding a sample diluent or a culture medium into the BD tube until the total volume is 9.7mL, adding collagenase, dispase and three antibodies into the BD tube after adding the sample diluent or the culture medium, wherein the adding amount of the collagenase, the dispase and the three antibodies is equal, the collagenase is collagenase-II, and the dispase is dispase-II.
2. The method for separating spleen macrophages of grass carp according to claim 1, wherein collagenase, dispase and tri-antibody are added in an amount of 100uL.
3. The method for separating grass carp spleen macrophages according to claim 1, wherein the digestion is performed in a water bath shaker at a water bath temperature of 30 ℃, comprising:
after addition, the mixture was digested for 40-50min in a water bath shaker at 30℃and the shaker rotation speed was 100rpm.
4. The method for separating grass carp spleen macrophages according to claim 1, wherein the digestion product is subjected to filtration and centrifugation, resulting in a filtered and centrifuged product, comprising:
the digestion products are filtered through a 200-mesh cell screen to obtain filtered grass carp spleen cell suspension, the filtered grass carp spleen cell suspension is resuspended and then transferred into a BD tube, and the cell suspension is obtained by supplementing 20mL with sample diluent and uniformly mixing;
adding the cell suspension into two BD tubes on average, pre-filling 10mL of lymphocyte separation liquid in each BD tube, balancing after adding, centrifuging for the first time after balancing, and taking out substances in critical layers of the lymphocyte separation liquid and the diluent after centrifuging to obtain a near white flocculent cell layer on the upper layer of the lymphocyte separation liquid;
and adding all the near white flocculent cell layers on the upper layer of the lymphocyte separation liquid into a BD tube pre-filled with sample diluent, carrying out balancing operation after adding, carrying out secondary centrifugation after balancing, removing supernatant in the BD tube after centrifugation, and carrying out resuspension after removal to obtain a product after filtration and centrifugation.
5. The method for separating grass carp spleen macrophages according to claim 4, wherein during the first centrifugation, the grass carp spleen macrophages are centrifuged for 15 minutes at a temperature of 22 ℃ and a rotation speed of 2950rpm using a basket centrifuge; at the said
In the second centrifugation, the basket centrifuge was used to centrifuge for 2 minutes at a temperature of 22℃and a rotational speed of 2390 rpm.
6. The method for separating spleen macrophages of grass carp according to claim 1, wherein the spleen macrophages of grass carp are obtained by culturing the product obtained after the filtering and centrifuging treatment by counting and counting the plates, comprising:
counting and plating the filtered and centrifuged products, plating the products, placing the products into a cell incubator for culturing to obtain macrophages, and performing cell cleaning treatment after culturing to obtain adherent macrophages;
observing whether the adherent macrophages meet the requirement of continuous culture, and if so, continuously culturing by using a culture medium to obtain the spleen macrophages of the grass carp.
7. The method of claim 6, wherein the medium is RPMI1640 medium containing 10% fbs and 1% tri-antibody.
8. The method for separating spleen macrophages of grass carp according to claim 6, wherein if the conditions are met, the culture medium is used for further culturing for at least 2 hours, so that the spleen macrophages of grass carp are obtained.
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