CN111961649A - Rat macrophage culture method - Google Patents
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- 210000000689 upper leg Anatomy 0.000 claims abstract description 24
- 239000001963 growth medium Substances 0.000 claims abstract description 23
- 239000007788 liquid Substances 0.000 claims abstract description 21
- 210000001185 bone marrow Anatomy 0.000 claims abstract description 19
- 210000003743 erythrocyte Anatomy 0.000 claims abstract description 19
- 238000007664 blowing Methods 0.000 claims abstract description 18
- 239000006166 lysate Substances 0.000 claims abstract description 17
- 210000003205 muscle Anatomy 0.000 claims abstract description 12
- 239000006228 supernatant Substances 0.000 claims abstract description 11
- 230000008569 process Effects 0.000 claims abstract description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 6
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- 210000003141 lower extremity Anatomy 0.000 claims abstract description 6
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- 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
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The invention discloses a rat macrophage culture method, which comprises the following steps: killing SD young rats born for 3 weeks by neck-removing method, soaking the rats in 75% alcohol for 3min, sterilizing, transferring to an ultra-clean workbench, cutting skin of hind limb, removing muscle of leg to expose femur, peeling off external muscle of femur, and cutting femur; cutting off femoral heads at two ends of a femur, sucking a certain amount of MEM-alpha basal medium liquid by using an injector, and blowing out bone marrow, wherein the bone marrow is washed clean in the blowing process; blowing and beating the bone marrow for 30 times by using a Pasteur pipette to prepare single cell suspension, centrifuging by using a centrifugal machine, collecting cells, and removing supernatant; add erythrocyte lysate. The invention carries out primary centrifugal separation, erythrocyte lysis, secondary centrifugal separation and special culture medium for macrophage on rat marrow, so that the cultured macrophage has higher purity and higher culture speed, and meets the research requirement.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a rat macrophage culture method.
Background
Macrophages are leukocytes in tissues and are derived from monocytes, the monocytes are derived from precursor cells in bone marrow, the macrophages and the monocytes are phagocytic cells, non-specific defense and specific defense are involved in vertebrates, the main functions of the macrophages and the monocytes are phagocytic to cell debris and pathogens in the form of fixed cells or free cells, lymphocytes or other immune cells are activated to react to the pathogens, and the macrophages belong to immune cells and have multiple functions, and are important objects for researching phagocytosis, cellular immunity and molecular immunology.
Disclosure of Invention
The invention aims to provide a rat macrophage culture method to solve the problems that the macrophage cultured by the existing rat macrophage culture method in the background art is low in purity, slow in growth cycle and not beneficial to experimental research.
In order to achieve the purpose, the invention provides the following technical scheme: a rat macrophage culturing method, comprising the steps of:
s1: killing SD young rats born for 3 weeks by neck-removing method, soaking the rats in 75% alcohol for 3min, sterilizing, transferring to an ultra-clean workbench, cutting skin of hind limb, removing muscle of leg to expose femur, peeling off external muscle of femur, and cutting femur;
s2: cutting off femoral heads at two ends of a femur, sucking a certain amount of MEM-alpha basal medium liquid by using an injector, and blowing out bone marrow, wherein the bone marrow is washed clean in the blowing process;
s3: blowing and beating the bone marrow for 30 times by using a Pasteur pipette to prepare single cell suspension, centrifuging by using a centrifugal machine, collecting cells, and removing supernatant;
s4: adding erythrocyte lysate, lysing for 3min at 4 ℃, centrifuging by a centrifuge, collecting cells, removing supernatant, adding macrophage special culture medium for re-suspending cell sap, and cleaning to remove residual erythrocyte lysate;
s5: the cell liquid is re-suspended by a special macrophage culture medium, the cells are inoculated into a T25 bottle for culture, non-adherent cells are transferred into a new T25 bottle for culture after 12 hours, the first liquid change is carried out after 48 hours, then the liquid change is carried out every 3 days, and the cultured macrophages can be harvested after the culture is carried out for the tenth day.
Preferably, the specific operation method of the necking method in S1 is set as follows: the neck of the rat is clamped by forceps with the left hand, the tail of the rat is grasped by the right hand, and the cervical vertebrae of the rat are pulled off by the two hands simultaneously.
Preferably, the rotation speed of the centrifuge in the S3 is set to 1000rmp, and the centrifugation time is set to 5 min.
Preferably, the rotation speed of the centrifuge in the S4 is set to 1000rmp, and the centrifugation time is set to 5 min.
Preferably, the preparation method of the erythrocyte lysate in S4 is set as follows: dissolving NH4Cl, KHCO3 and Na2EDTA in deionized water respectively, stirring uniformly, adding deionized water to a constant volume to a required volume, adjusting the pH value of the solution to 7.2-7.4, filtering with a 0.22um filter, and storing at 2-8 ℃ for later use.
Preferably, the preparation method of the special culture medium for macrophages in S4 and S5 is as follows: taking a certain amount of MEM-alpha basic culture medium in a clean bottle, adding FBS, 50U/ml penicillin, 100U/ml streptomycin and 30ng/ml CSF, filtering by a 0.22um filter, transferring to a new bottle, and storing in a refrigerator at 4 ℃ for later use.
Compared with the prior art, the invention has the beneficial effects that.
(1) The invention greatly improves the purity of the prepared macrophage by carrying out primary centrifugal separation, red blood cell lysis and secondary centrifugal separation on the rat bone marrow cells, and the utilization rate of the rat bone marrow cells is higher.
(2) The invention greatly shortens the culture period of the macrophage and improves the culture success rate of the macrophage by preparing the special culture medium for the macrophage.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides the following examples:
example 1
A method for culturing rat macrophages, the method comprising the steps of:
s1: killing SD young rats born for 3 weeks by neck-removing method, soaking the rats in 75% alcohol for 3min, sterilizing, transferring to an ultra-clean workbench, cutting skin of hind limb, removing muscle of leg to expose femur, peeling off external muscle of femur, and cutting femur;
s2: cutting off femoral heads at two ends of a femur, sucking the MEM-alpha basal medium by using a 5ml syringe, and blowing out bone marrow, wherein the bone marrow is washed clean in the blowing process;
s3: blowing and beating the bone marrow for 30 times by using a Pasteur pipette to prepare single cell suspension, centrifuging by using a centrifugal machine, collecting cells, and removing supernatant;
s4: adding 3ml of erythrocyte lysate, lysing for 3min at 4 ℃, centrifuging by a centrifuge, collecting cells, removing supernatant, adding 2ml of macrophage-dedicated culture medium basic suspension cell liquid, and cleaning to remove residual erythrocyte lysate;
s5: the cell liquid is re-suspended by 5ml of special macrophage culture medium, the cells are inoculated into a T25 bottle for culture, non-adherent cells are transferred into a new T25 bottle for culture after 12h, the first liquid change is carried out after 48h, then the liquid change is carried out every 3 days, and the cultured macrophages can be harvested after the culture is carried out for the tenth day.
Further, the specific operation method of the neck removal method in S1 is set as follows: the neck of the rat is clamped by forceps with the left hand, the tail of the rat is grasped by the right hand, and the cervical vertebrae of the rat are pulled off by the two hands simultaneously.
Further, the rotation speed of the centrifuge in S3 is set to 1000rmp, and the centrifugation time is set to 5 min.
Further, the rotation speed of the centrifuge in S4 is set to 1000rmp, and the centrifugation time is set to 5 min.
Further, the preparation method of the erythrocyte lysate in the S4 is set as follows: dissolving NH4Cl 80g, KHCO 310 g and Na2EDTA 3.7g in 900ml deionized water respectively, stirring, adding deionized water to reach volume of 1000ml, adjusting pH value of the solution to 7.2-7.4, filtering with 0.22um filter, and storing at 2-8 deg.C for later use.
Further, the preparation method of the special culture medium for macrophages in S4 and S5 is set as follows: 85ml of MEM-alpha basic culture medium is taken from a clean bottle with a sterile base in a clean bench, 15ml of FBS, 50U/ml of penicillin, 100U/ml of mitomycin and 30ng/ml of CSF are added, a 0.22um filter is used for filtering, and then the mixture is transferred to a new bottle and is stored in a refrigerator at 4 ℃ for later use.
Example 2
A method for culturing rat macrophages, the method comprising the steps of:
s1: killing SD young rats born for 3 weeks by neck-removing method, soaking the rats in 75% alcohol for 3min, sterilizing, transferring to an ultra-clean workbench, cutting skin of hind limb, removing muscle of leg to expose femur, peeling off external muscle of femur, and cutting femur;
s2: cutting off femoral heads at two ends of a femur, sucking the MEM-alpha basal medium by using a 10ml syringe, blowing out bone marrow, and washing the bone marrow in the blowing process;
s3: blowing and beating the bone marrow for 30 times by using a Pasteur pipette to prepare single cell suspension, centrifuging by using a centrifugal machine, collecting cells, and removing supernatant;
s4: adding 6ml of erythrocyte lysate, lysing for 3min at 4 ℃, centrifuging by a centrifuge, collecting cells, removing supernatant, adding 4ml of special macrophage culture medium for re-suspending cell lysate, and cleaning to remove residual erythrocyte lysate;
s5: the cell liquid is re-suspended by 10ml of special macrophage culture medium, the cells are inoculated into a T25 bottle for culture, non-adherent cells are transferred into a new T25 bottle for culture after 12h, the first liquid change is carried out after 48h, then the liquid change is carried out every 3 days, and the cultured macrophages can be harvested after the culture is carried out for the tenth day.
Further, the specific operation method of the neck removal method in S1 is set as follows: the neck of the rat is clamped by forceps with the left hand, the tail of the rat is grasped by the right hand, and the cervical vertebrae of the rat are pulled off by the two hands simultaneously.
Further, the rotation speed of the centrifuge in S3 is set to 1000rmp, and the centrifugation time is set to 5 min.
Further, the rotation speed of the centrifuge in S4 is set to 1000rmp, and the centrifugation time is set to 5 min.
Further, the preparation method of the erythrocyte lysate in the S4 is set as follows: dissolving NH4Cl 80g, KHCO 310 g and Na2EDTA 3.7g in 900ml deionized water respectively, stirring, adding deionized water to reach volume of 1000ml, adjusting pH value of the solution to 7.2-7.4, filtering with 0.22um filter, and storing at 2-8 deg.C for later use.
Further, the preparation method of the special culture medium for macrophages in S4 and S5 is set as follows: 85ml of MEM-alpha basic culture medium is taken from a clean bottle with a sterile base in a clean bench, 15ml of FBS, 50U/ml of penicillin, 100U/ml of mitomycin and 30ng/ml of CSF are added, a 0.22um filter is used for filtering, and then the mixture is transferred to a new bottle and is stored in a refrigerator at 4 ℃ for later use.
Example 3
A method for culturing rat macrophages, the method comprising the steps of:
s1: killing SD young rats born for 3 weeks by neck-removing method, soaking the rats in 75% alcohol for 3min, sterilizing, transferring to an ultra-clean workbench, cutting skin of hind limb, removing muscle of leg to expose femur, peeling off external muscle of femur, and cutting femur;
s2: cutting off femoral heads at two ends of a femur, sucking the MEM-alpha basal medium by using a 15ml syringe, and blowing out bone marrow, wherein the bone marrow is washed clean in the blowing process;
s3: blowing and beating the bone marrow for 30 times by using a Pasteur pipette to prepare single cell suspension, centrifuging by using a centrifugal machine, collecting cells, and removing supernatant;
s4: adding 9ml of erythrocyte lysate, lysing for 3min at 4 ℃, centrifuging by a centrifuge, collecting cells, removing supernatant, adding 6ml of special macrophage culture medium for re-suspending cell lysate, and cleaning to remove residual erythrocyte lysate;
s5: the cell liquid is re-suspended by using 15ml of special macrophage culture medium, the cells are inoculated into a T25 bottle for culture, nonadherent cells are transferred into a new T25 bottle for culture after 12h, the liquid is changed for the first time after 48h, then the liquid is changed every 3 days, and the cultured macrophages can be harvested after the culture is carried out for the tenth day.
Further, the specific operation method of the neck removal method in S1 is set as follows: the neck of the rat is clamped by forceps with the left hand, the tail of the rat is grasped by the right hand, and the cervical vertebrae of the rat are pulled off by the two hands simultaneously.
Further, the rotation speed of the centrifuge in S3 is set to 1000rmp, and the centrifugation time is set to 5 min.
Further, the rotation speed of the centrifuge in S4 is set to 1000rmp, and the centrifugation time is set to 5 min.
Further, the preparation method of the erythrocyte lysate in the S4 is set as follows: dissolving NH4Cl 80g, KHCO 310 g and Na2EDTA 3.7g in 900ml deionized water respectively, stirring, adding deionized water to reach volume of 1000ml, adjusting pH value of the solution to 7.2-7.4, filtering with 0.22um filter, and storing at 2-8 deg.C for later use.
Further, the preparation method of the special culture medium for macrophages in S4 and S5 is set as follows: 85ml of MEM-alpha basic culture medium is taken from a clean bottle with a sterile base in a clean bench, 15ml of FBS, 50U/ml of penicillin, 100U/ml of mitomycin and 30ng/ml of CSF are added, a 0.22um filter is used for filtering, and then the mixture is transferred to a new bottle and is stored in a refrigerator at 4 ℃ for later use.
The working principle is as follows: through carrying out centrifugation once to rat marrow, collect the cell, abundant schizolysis is carried out to rethread erythrocyte lysate, carry out secondary centrifugation through centrifuge again at last, make the stock cell in the rat marrow can obtain make full use of, and multiple separation makes the purity of cell obtain improving greatly, and then make the purity of the macrophage that makes obtain very big improvement, through preparing special culture medium of macrophage, make the culture cycle of macrophage shorten greatly, and the cultivation success rate of macrophage also obtains improving, it is once to change the liquid every 3 days, through continuous liquid changing operation, ensure that the cell nutrition of cultivation is abundant, improve the cultivation success rate, shorten the culture cycle.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. The rat macrophage culture method is characterized by comprising the following steps: the method comprises the following steps:
s1: killing SD young rats born for 3 weeks by neck-removing method, soaking the rats in 75% alcohol for 3min, sterilizing, transferring to an ultra-clean workbench, cutting skin of hind limb, removing muscle of leg to expose femur, peeling off external muscle of femur, and cutting femur;
s2: cutting off femoral heads at two ends of a femur, sucking a certain amount of MEM-alpha basal medium liquid by using an injector, and blowing out bone marrow, wherein the bone marrow is washed clean in the blowing process;
s3: blowing and beating the bone marrow for 30 times by using a Pasteur pipette to prepare single cell suspension, centrifuging by using a centrifugal machine, collecting cells, and removing supernatant;
s4: adding erythrocyte lysate, lysing for 3min at 4 ℃, centrifuging by a centrifuge, collecting cells, removing supernatant, adding macrophage special culture medium for re-suspending cell sap, and cleaning to remove residual erythrocyte lysate;
s5: the cell liquid is re-suspended by a special macrophage culture medium, the cells are inoculated into a T25 bottle for culture, non-adherent cells are transferred into a new T25 bottle for culture after 12 hours, the first liquid change is carried out after 48 hours, then the liquid change is carried out every 3 days, and the cultured macrophages can be harvested after the culture is carried out for the tenth day.
2. The rat macrophage culturing method according to claim 1, wherein: the specific operation method of the neck removing method in the S1 is as follows: the neck of the rat is clamped by forceps with the left hand, the tail of the rat is grasped by the right hand, and the cervical vertebrae of the rat are pulled off by the two hands simultaneously.
3. The rat macrophage culturing method according to claim 1, wherein: the rotating speed of the centrifuge in the S3 is set to be 1000rmp, and the centrifugation time is set to be 5 min.
4. The rat macrophage culturing method according to claim 1, wherein: the rotating speed of the centrifuge in the S4 is set to be 1000rmp, and the centrifugation time is set to be 5 min.
5. The rat macrophage culturing method according to claim 1, wherein: the preparation method of the erythrocyte lysate in the S4 is as follows: dissolving NH4Cl, KHCO3 and Na2EDTA in deionized water respectively, stirring uniformly, adding deionized water to a constant volume to a required volume, adjusting the pH value of the solution to 7.2-7.4, filtering with a 0.22um filter, and storing at 2-8 ℃ for later use.
6. The rat macrophage culturing method according to claim 1, wherein: the preparation method of the culture medium special for the macrophages in the S4 and the S5 is as follows: taking a certain amount of MEM-alpha basic culture medium in a clean bottle, adding FBS, 50U/ml penicillin, 100U/ml streptomycin and 30ng/ml CSF, filtering by a 0.22um filter, transferring to a new bottle, and storing in a refrigerator at 4 ℃ for later use.
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| CN114574439A (en) * | 2022-01-29 | 2022-06-03 | 国药集团动物保健股份有限公司 | Preparation method of pig bone marrow cells and pig cancellous bone crushing device |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN114574439A (en) * | 2022-01-29 | 2022-06-03 | 国药集团动物保健股份有限公司 | Preparation method of pig bone marrow cells and pig cancellous bone crushing device |
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