CN114438029A - Method for sorting intratesticular macrophages - Google Patents
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Abstract
The invention discloses a sorting method of intratesticular macrophages, and relates to the technical field of biological cells. The invention provides application of CD74 as a protein marker in macrophage classification. Through a large amount of experimental screening, the inventor finally finds that the testis macrophage protein marker CD74 has wider expression in a testis macrophage group and embodies the strongest testis specificity compared with the existing common macrophage protein markers including F4/80, CD206, CD86, CD68, CD14, CD163 and CD11 b; the method for sorting the macrophages in the testis can accurately and efficiently separate the macrophages in the testis.
Description
Technical Field
The invention relates to the technical field of biological cells, in particular to a method for sorting intratesticular macrophages.
Background
Macrophages are immune cells and are part of the mononuclear phagocyte system, whose main role is to phagocytize dead cells, cell debris, pathogens and other foreign bodies. In addition, they also have signal transduction and regulatory functions. Macrophages have great tissue specificity and plasticity and are capable of changing their phenotype in response to environmental signals. At present, macrophages have been extensively and intensively studied in the fields of aging, immunity, development, tissue repair, cancer, and the like. Macrophages are present in many tissues of the human body and are not a functionally uniform population of cells, but rather a population of cells with a rich heterogeneity. Different macrophage subsets have different gene expression profiles and cellular functions. To better understand these differences, flow sorting studies are currently mainly performed on different protein markers on the macrophage surface. Common macrophage protein markers include: f4/80, CD206, CD86, CD68, CD14, CD163, CD11b and the like.
With the continuous development of single cell sequencing technology, more and more macrophage surface protein markers are discovered, and this also enables more and more macrophage subpopulations in different tissues to be explored, such as tumor-associated TCR+CD169 related to macrophage and erythropoiesis+Macrophage and chronic heart failure associated CD72+The macrophage cell of (1). However, for testis macrophages, the labeling is still mainly carried out according to surface protein markers F4/80, CD11b and the like reported by other tissues, and the characteristic of strong tissue specificity of the macrophages is ignored, so that more functional testis macrophage subgroups are difficult to deeply research.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for sorting intratesticular macrophages.
In order to achieve the purpose, the invention adopts the technical scheme that: use of CD74 as a protein marker in macrophage classification.
The inventor of the application aims at the defect that the tissue specificity of the existing intratesticular macrophage surface protein marker is not strong, combines a single cell sequencing technology, takes a mouse as a research object, analyzes the surface protein marker of the intratesticular macrophage of the mouse under different age states, and screens out the marker with the strongest tissue specificity and the best sorting effect, thereby constructing a novel method for sorting the intratesticular macrophage.
As a preferred embodiment of the use of CD74 as a protein marker in the sorting of macrophages, the macrophages are intratesticular macrophages.
The invention also provides a sorting method of the intratesticular macrophages, which is used for sorting testicular tissue cells through the CD74 antibody to obtain the intratesticular macrophages.
As a preferred embodiment of the method for sorting intratesticular macrophages according to the present invention, the method for sorting intratesticular macrophages comprises the steps of:
(1) obtaining testis tissue pulp;
(2) obtaining testis tissue cells;
(3) CD74 antibody incubated testis tissue cells;
(4) testicular macrophages were obtained using flow assay detection.
As a preferred embodiment of the method for sorting intratesticular macrophages according to the present invention, the method for sorting intratesticular macrophages specifically comprises the steps of:
(1) taking testis, removing testis white membrane, adding PBS, and cutting testis into paste to obtain testis tissue slurry;
(2) taking the testis tissue pulp, adding type IV collagenase, digesting in water bath at 37 ℃ for 15min, blowing and beating the testis tissue pulp, adding ice PBS to stop digestion, filtering by a 70-micron screen, centrifuging the cell suspension at 1500rpm for 5min, taking cell precipitate to obtain testis tissue cells;
(3) adding the testis tissue cells into PBS for resuspension, taking the cell suspension, adding the cell suspension into a flow tube, and adding a PBS solution containing 2% FBS as a negative control sample; then adding the CD74 antibody into the cell suspension for dyeing, and incubating for 30min at 4 ℃ in the dark; after dyeing is finished, centrifuging at 1500rpm for 5min, and taking cell sediment; resuspending the cell pellet with PBS, centrifuging at 1500rpm for 5min, discarding the supernatant, and repeating twice; adding a PBS solution containing 2% FBS into the cell sediment for resuspension, filtering by a 40-micron screen, and reserving filtrate to obtain cell suspension;
(4) and (4) carrying out flow sorting on the cell suspension in the step (3) to obtain the macrophage in the pill.
Through a large number of experiments, the inventor of the application finds that when testis tissue pulp is obtained, the testis tissue is cut into paste, and the operation time is controlled within 3-5min, so that the influence on the activity of cells can be avoided.
As a preferred embodiment of the method for sorting the intratesticular macrophages, the CD74 antibody is present in a concentration of less than 0.5. mu.g/million cell.
In a preferred embodiment of the method for sorting intratesticular macrophages according to the present invention, the collagenase type IV concentration is 1 mg/mL.
Through a large number of experiments, the inventor of the application finds that when the collagenase type IV is used for digesting testis tissues, the digestion effect is the best when the collagenase type IV is at a concentration of 1 mg/mL.
The invention also provides the intratesticular macrophage prepared by the method for sorting the intratesticular macrophage.
The invention also provides application of the intratesticular macrophages in functional research of the intratesticular macrophages. At present, testis macrophages are marked mainly according to surface protein markers F4/80, CD11b and the like reported by other tissues, but the characteristic of strong tissue specificity of the macrophages is ignored, so that more functional intratestis macrophage subgroups are difficult to deeply research. The selected intratesticular macrophages adopt the protein marker CD74, the sorting effect is good, and the method can be used for research related to testicular macrophages.
The invention also provides a kit for sorting intratesticular macrophages, the kit comprising a CD74 antibody.
The invention has the beneficial effects that: the invention provides a sorting method of intratesticular macrophages, and the inventor of the application finally discovers that compared with the existing common macrophage protein markers, the testis macrophage protein marker CD74 provided by the invention comprises F4/80, CD206, CD86, CD68, CD14, CD163 and CD11b, has wider expression in testis macrophage groups and shows the strongest testis specificity through a large amount of experimental screening; the method for sorting the macrophages in the testis can accurately and efficiently separate the macrophages in the testis.
Drawings
FIG. 1 is a graph of intratesticular cell distribution in 3 month old (3M) and 21 month old (21M) mice.
FIG. 2 is a graph of total cell populations in testis from 3-month-old (3M) and 21-month-old (21M) mice.
FIG. 3 is a heat map of specific cell markers in mouse testicular single cell sequencing and a functional analysis thereof.
FIG. 4 is a graph of macrophage marker expression in testis single cell sequencing for 3 month old (3M) and 21 month old (21M) mice.
FIG. 5 is a graph showing CD74 expression of testis CD74 positive macrophages from 3 (3M) and 21 (21M) months old mice after flow sorting.
FIG. 6 shows the expression of common macrophage protein markers in normal transcriptome sequencing after flow sorting of 3-month-old (3M) and 21-month-old (21M) mouse testis CD74 positive macrophages.
FIG. 7 is a diagram of cell function analysis of common transcriptome sequencing after flow sorting of Cd74 positive macrophages from 3-month (3M) and 21-month (21M) mouse testes.
Detailed Description
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Example 1
The embodiment provides a method for sorting intratesticular macrophages, and the method is adopted to sort intratesticular macrophages of mice with 3 months of age (3M) and 21 months of age (21M), and the specific experimental method is as follows:
(1) obtaining testis tissue pulp: killing the mouse by dislocation of cervical vertebra, dissecting the abdominal cavity, removing redundant fat, and taking out testis tissue; after removing the white membrane of the testis, adding a proper amount of PBS, and shearing the testis tissue into paste by using a pair of micro scissors to obtain testis tissue slurry;
(2) obtaining testis tissue cells: adding 1mL of 1mg/mL IV type collagenase into bilateral testis of each mouse, digesting in water bath at 37 ℃ for 15min, blowing for about 7min to obtain flocculent shape, and beating once; adding 6mL of ice PBS to terminate digestion, filtering by a 70-micron screen, placing the cell suspension at 1500rpm, centrifuging for 5min, and taking a precipitate to obtain testis tissue cells;
(3) CD74 antibody incubated testicular tissue cells: adding 1mL of PBS into the testis tissue cells for resuspension, adding 200 mu L of cell suspension into a flow tube, and adding 800 mu L of PBS solution containing 2% FBS as a negative control sample; adding Alexa Fluor647anti-mouse CD74 (antibody concentration is less than 0.5 mu g/million cell) into the residual cell suspension for staining, and incubating at 4 ℃ for 30min in the dark; after dyeing is finished, centrifuging the mixed solution for 5min at 1500rpm, and removing supernatant; resuspending the cell pellet with PBS, centrifuging at 1500rpm for 5min, discarding the supernatant, and repeating twice to remove residual antibody; finally, adding 2mL of PBS solution containing 2% FBS into the cell sediment for resuspension, and filtering the cell sediment by using a 40-micron screen to remove redundant impurities to obtain cell suspension;
(4) testicular macrophages were obtained using flow assay detection.
Example 2
In this example, 3 month old (3M) and 21 month old (21M) mouse testis tissue cells extracted in example 1 were subjected to cell sequencing, and distribution of cells in the testis, total cell grouping in the testis, and expression of testis cell macrophage marker in mice of different ages were observed. The results of the experiment are shown in FIGS. 1 to 4. As can be seen from fig. 1, 7524 testicular cells were captured in the 3M group, and 3597 testicular cells were captured in the 21M group. The ratio of cell numbers between the two did not change significantly. As can be seen from fig. 2, after removing the haploid, the main cell populations in 3M and 21M testicles include intrinsic lymphocytes, pericyte-like cells, endothelial cells, leydig cells, macrophages, spermatogonial cells, spermatocytes, long sperm, round sperm and supporting cells. In addition, the results better reduce the overall process of spermatogenesis. The result of the testicular single cell sequencing of FIG. 3 truly reduced the function of the different cells in the testis and their specific cell markers, including the antigen presentation function of macrophages and their markers Cd74 and F4/80. As can be seen from FIG. 4, Cd74 has a wider and higher level expression in testis macrophage population than the existing common macrophage protein markers F4/80, Cd206, Cd86, Cd68, Cd14, Cd163, Cd11b and the like.
Example 3
In this example, 3 month-old (3M) and 21 month-old (21M) mouse testis macrophages extracted in example 1 were subjected to cell sequencing, and the expression of macrophage CD74 and each protein marker in mice of different ages was observed. The results of the experiment are shown in FIGS. 5 to 7. As is clear from fig. 5 to 7, the expression level of CD74 was higher in testicular macrophages of mice aged 3 months (3M) and 21 months (21M) than other proteins.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
- Use of CD74 as a protein marker in macrophage sorting.
- 2. The use of claim 1, wherein the macrophage is an intratesticular macrophage.
- 3. A method for sorting intratesticular macrophages is characterized in that testicular tissue cells are sorted by a CD74 antibody to obtain the intratesticular macrophages.
- 4. The method of sorting intratesticular macrophages of claim 3, comprising the steps of:(1) obtaining testis tissue pulp;(2) obtaining testis tissue cells;(3) CD74 antibody incubated testis tissue cells;(4) testicular macrophages were obtained using flow assay detection.
- 5. The method for sorting intratesticular macrophages according to claim 3, comprising the steps of:(1) taking testis, removing testis white membrane, adding PBS, and cutting testis into paste to obtain testis tissue slurry;(2) taking the testis tissue pulp, adding type IV collagenase, digesting in water bath at 37 ℃ for 15min, blowing and beating the testis tissue pulp, adding ice PBS to stop digestion, filtering by a 70-micron screen, centrifuging the cell suspension at 1500rpm for 5min, and taking cell precipitate to obtain testis tissue cells;(3) adding the testis tissue cells into PBS for resuspension, taking the cell suspension, adding the cell suspension into a flow tube, and adding a PBS solution containing 2% FBS as a negative control sample; then adding the CD74 antibody into the cell suspension for dyeing, and incubating for 30min at 4 ℃ in the dark; after dyeing is finished, centrifuging at 1500rpm for 5min, and taking cell sediment; resuspending the cell pellet with PBS, centrifuging at 1500rpm for 5min, discarding the supernatant, and repeating twice; adding a PBS solution containing 2% FBS into the cell sediment for resuspension, filtering by a 40-micron screen, and reserving filtrate to obtain cell suspension;(4) and (4) carrying out flow sorting on the cell suspension in the step (3) to obtain the macrophage in the pill.
- 6. The method for sorting intratesticular macrophages according to claim 5, wherein the concentration of said CD74 antibody is less than 0.5 μ g/million cell.
- 7. The method for sorting intratesticular macrophages according to claim 5, wherein the collagenase type IV is present in a concentration of 1 mg/mL.
- 8. An intratesticular macrophage prepared according to the method of any one of claims 3-7.
- 9. Use of intratesticular macrophages according to claim 8 in the study of intratesticular macrophage functionality.
- 10. A kit for sorting intratesticular macrophages, said kit comprising a CD74 antibody.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111394302A (en) * | 2020-02-24 | 2020-07-10 | 中山大学 | Method for separating and culturing human testicular interstitial stem cells |
| CN112067820A (en) * | 2020-06-28 | 2020-12-11 | 暨南大学 | Application of CD74 protein in preparation of kit for identifying macrophage subset in brain after ischemic injury |
| WO2021231648A2 (en) * | 2020-05-12 | 2021-11-18 | Gigagen, Inc. | Cancer therapeutics comprising chemokine or its analog |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111394302A (en) * | 2020-02-24 | 2020-07-10 | 中山大学 | Method for separating and culturing human testicular interstitial stem cells |
| WO2021231648A2 (en) * | 2020-05-12 | 2021-11-18 | Gigagen, Inc. | Cancer therapeutics comprising chemokine or its analog |
| CN112067820A (en) * | 2020-06-28 | 2020-12-11 | 暨南大学 | Application of CD74 protein in preparation of kit for identifying macrophage subset in brain after ischemic injury |
Non-Patent Citations (1)
| Title |
|---|
| EUBLER K等: "Exploring the Ion Channel TRPV2 and Testicular Macrophages in Mouse Testis", 《INT J MOL SCI.》, vol. 22, no. 9, pages 3 * |
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