Method for separating and purifying aTreg cells in mouse body
By staining aTreg cells in mice with multiple markers and sorting methods, the problem of difficulty in isolating aTreg cells in the prior art was solved, and a high purity and high efficiency purification effect was achieved.
Patent Information
- Application Number
- CN202510396522.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-31
- Publication Date
- 2025-07-01
AI Technical Summary
The prior art is difficult to effectively isolate and purify activated-like Treg cells (aTreg cells) in mice. Due to their small differences from other regulatory T cells, it leads to difficulty in isolation and complicated steps.
By staining the markers such as CD4, Foxp3, CD44 and CD62L and sorting with flow cytometry, the operation is simplified and the extraction efficiency of aTreg cells is improved.
Surviving aTreg cells were successfully isolated and purified, with a purity of 95%-99%, which simplified the difficulty of operation and improved the extraction efficiency.
Smart Images
Figure CN120230714A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of cell sorting, and particularly to a method for separating and purifying aTreg cells in mice. Background Art
[0002] aTreg cells (activated Treg) are a subtype of regulatory T cells (Treg cells) - activated-like Treg cells. They have a strong immunosuppressive function, the ability to migrate into tissues, play an important inhibitory role in the inflammatory response in tissues, and play an important role in inhibiting immune activity and in autoimmune diseases.
[0003] However, in the research process, due to the small differences between aTreg cells and other types of regulatory T cells, it is difficult to separate aTreg cells from T cells, the extraction steps are complex, and it is difficult to extract viable aTreg cells. Summary of the Invention
[0004] In view of the deficiencies of the above-mentioned related technologies, this application provides a method for separating and purifying aTreg cells in mice. By staining multiple markers and then sorting by flow cytometry, while simplifying the operation, viable aTreg cells are separated and extracted under the combined action of multiple stained markers.
[0005] The method for separating and purifying aTreg cells in mice provided by this application adopts the following technical solution:
[0006] A method for separating and purifying aTreg cells in mice, comprising the following steps: sorting the stained mouse lymphocytes by flow cytometry, and the markers for staining include one or more of CD4, Foxp3, CD44, and CD62L.
[0007] Preferably, the markers for staining include CD4, Foxp3, CD44, and CD62L.
[0008] Preferably, the mouse lymphocytes are taken from a mouse with at least one marker of Foxp3 stained with fluorescence.
[0009] Preferably, the mouse lymphocytes are taken from a mouse with Foxp3 stained with fluorescence.
[0010] Preferably, CD4, CD44, and CD62L are stained by adding surface staining antibodies to the mouse lymphocytes.
[0011] Preferably, the surface staining antibodies for staining the CD4 include one or more of CD4-BV421, CD4-FITC, and CD4-APC.
[0012] Preferably, the surface staining antibody for staining the CD4 is CD4-BV421.
[0013] Preferably, the surface staining antibodies for staining the CD44 include one or more of CD44-PE-cy7, CD44-PE, and CD44-APC.
[0014] Preferably, the surface staining antibody for staining the CD44 is CD44-PE-cy7.
[0015] Preferably, the surface staining antibodies for staining the CD62L- include one or more of CD62L-PC5.5, CD62L-Vioblue, and CD62L-FITC.
[0016] Preferably, the surface staining antibody for staining the CD62L-PC5.5 is CD62L-5.5.
[0017] Preferably, the Foxp3 of the mouse is fluorescently labeled with one or two of YFP and GFP.
[0018] Preferably, the Foxp3 of the mouse is fluorescently labeled with YFP.
[0019] Preferably, the mouse lymphocytes are obtained by the following steps: taking mouse spleen and lymph node tissues, grinding them, filtering through a 40μm filter to obtain a single cell suspension, adding erythrocyte lysate for 35 - 45s, then adding PBS buffer to stop lysis, centrifuging at a speed of 1250 - 1350r / min for 5 minutes, discarding the supernatant, and washing twice with PBS buffer to obtain mouse lymphocytes.
[0020] Preferably, the purity of the isolated and purified aTreg cells reaches 95% - 99%.
[0021] In summary, the present application includes at least one of the following beneficial technical effects:
[0022] 1. In the present application, by staining multiple markers and then sorting through flow cytometry, under the combined action of multiple stained markers, viable aTreg cells are successfully isolated and purified from T cells in the mouse body;
[0023] 2. The method for isolating and purifying aTreg cells in the mouse body in the present application refers to Figure 2, from left to right, it is shown that the boundaries between lymphocytes and other cells in the extracted lymphocyte suspension are distinct, and the separation effect is remarkable. The boundaries between Treg cells and non-Treg cells in lymphocytes are distinct, and the separation effect is remarkable. The boundaries between the cells that simultaneously express CD4 and Foxp3 in Treg cells and other cells are distinct, and the separation effect is remarkable. The boundaries between aTreg cells (CD44+CD62L-) and non-aTreg cells in the cells that simultaneously express CD4 and Foxp3 are distinct, and the separation effect is remarkable;
[0024] 3. In this application, by staining multiple markers and then sorting through flow cytometry, on the one hand, viable aTreg cells that can be cultured can be directly obtained without breaking the cell membrane for staining. On the other hand, the operation difficulty is simplified, and the extraction efficiency of aTreg cells is improved. Brief Description of the Drawings
[0025] Figure 1 It is a comparison chart of the protein blot detection results of aTreg cells and non-aTreg cells in Example 1;
[0026] Figure 2 It is a comparison chart of the number of aTreg cells in Example 1. Detailed Embodiments
[0027] In order to make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be further described in detail below in conjunction with embodiments. The equipment and reagents used in each embodiment and test example can be obtained from commercial channels without special instructions. The specific embodiments described here are only used to explain the present invention and are not used to limit the present invention.
[0028] Example 1
[0029] The specific steps of a method for separating and purifying aTreg cells in mice provided in Example 1 of this application are as follows:
[0030] (1) Lymphocyte extraction: After grinding the spleen and lymph node tissues of mice, a single cell suspension is obtained by filtering through a 40μm filter. Add erythrocyte lysate for 40s, and then add PBS to stop lysis. Centrifuge at a speed of 1300r / min for 5 minutes to discard the supernatant, and wash twice with PBS buffer to obtain a mouse lymphocyte suspension.
[0031] (2) Sorting of aTreg cells: Add surface staining antibodies CD4-BV421 (BD, 562891), CD44-PE-cy7 (BD, 560569), and CD62L-5.5 (BD, 560513) to the mouse lymphocyte suspension for flow cytometry staining and incubate at 4 °C for 30 minutes. Use a flow cytometry cell sorter to sort non-aTreg cells (CD4+YFP-) and aTreg cells (CD4+YFP+CD44+CD62L-) from the mouse. Resuspend the sorted aTreg cells at a concentration of 5×10 5 cells / ml in complete T cell medium (1640, 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, 5 μM L-glutamine, and 1×HEPES).
[0032] The mice in this example are B6.129(Cg)-Foxp3tm4(YFP / icre)Ayr / J (Stock No: 016959) mice, purchased from Jackson Laboratory. The mice are housed in a specific pathogen-free (SPF level) environment in the Animal Experiment Center of Renmin Hospital of Wuhan University, with free access to water and food, at a temperature of 18 °C - 22 °C and moderate humidity. All experimental operations have obtained the permission of the Animal Ethics Committee of Renmin Hospital of Wuhan University (Permit No.: WDRM20170310), and its Foxp3 is fluorescently labeled with YFP.
[0033] Experimental detection
[0034] (1) The detection of Foxp3 expression was performed on the aTreg cells and non-aTreg cells obtained by using the method for separating and purifying aTreg cells in mice in Example 1. The specific steps are as follows: After centrifuging the cells, the supernatant was discarded, and RIPA lysis buffer (containing phosphatase inhibitor and protease inhibitor at 2 mg / ml) was added. After cell lysis was completed, 4× protein loading buffer was added, and the mixture was boiled at 95 °C for 10 minutes. The extracted proteins were electrophoretically separated in an SDS-PAGE gel, and then the proteins separated by electrophoresis were transferred onto a PVDF membrane (GE Life, USA, #10600023). After the transfer was completed, the membrane was placed in 5% skim milk for blocking for 1 hour. The blocked membrane was washed 3 times with TBS-Tween20, and then sequentially placed in the corresponding primary antibodies Foxp3 (Servicebio, GB112325-100) and GAPDH (Servicebio, GB15002-100) and incubated overnight at 4 °C. The concentrations of these antibodies were prepared according to the instructions. The next day, the incubated membrane was washed 3 times with TBS-Tween20. After washing was completed, it was placed in a chemical secondary antibody (1:20000) and incubated at room temperature for 1 hour. After incubation was completed, it was washed 3 times with TBS-Tween20 again. The washed membrane was placed in ECL luminescent solution for activation, and the activated membrane was placed in an imager for development, and the expression results were obtained as Figure 1 shown, where the result of aTreg cells was labeled as aTreg CD4+YFP+CD44+CD62L-, and the result of non-aTreg cells was labeled as No-aTreg CD4+YFP-.
[0035] (2) The comparison chart of the number of aTreg cells obtained by using the method for separating and purifying aTreg cells in mice in Example 1 is as Figure 2 , and the sorting purity of aTreg cells reached more than 98%.
[0036] Result analysis The following is a detailed description of the present application in combination with Figure 1-2 the experimental results provided.
[0038] Referring to Figure 1 it can be seen that the expression level of Foxp3 in the aTreg cell group obtained by using the method for separating and purifying aTreg cells in mice in Example 1 was significantly higher than that in the non-aTreg cell group, verifying that the method for separating and purifying aTreg cells in mice in Example 1 can effectively sort out the important subtype aTreg cells in Treg cells.
[0039] Referring to Figure 2, it is described in turn from left to right that the boundaries between lymphocytes and other cells in the extracted lymphocyte suspension are distinct and the separation effect is remarkable, the boundaries between Treg cells and non-Treg cells in lymphocytes are distinct and the separation effect is remarkable, the boundaries between cells that simultaneously express CD4 and Foxp3 in Treg cells and other cells are distinct and the separation effect is remarkable, and the boundaries between aTreg cells (CD44+CD62L-) and non-aTreg cells in cells that simultaneously express CD4 and Foxp3 are distinct and the separation effect is remarkable. The proportion of aTreg cells is 60.5%, and the sorting purity is greater than 98%. In summary, the method for separating and purifying aTreg cells in mice in this application can extract viable aTreg cells that can be cultured without breaking the cell membrane for staining, with remarkable separation and purification effects, simple operation, and high separation efficiency.
[0040] This specific embodiment is only an interpretation of this application, and it is not a limitation to this application. Those skilled in the art can make modifications without creative contributions to this embodiment according to needs after reading this specification, but as long as it is within the scope of the claims of this application, it is protected by the patent law.
Claims
1. A method for separating and purifying aTreg cells in mice, characterized in that: It includes the following steps: sorting the stained mouse lymphocytes by flow cytometry, and the markers for the staining include one or more of CD4, Foxp3, CD44 and CD62L.
2. The method for separating and purifying aTreg cells in mice according to claim 1, wherein: The markers for the staining include CD4, Foxp3, CD44 and CD62L.
3. The method for separating and purifying aTreg cells in a mouse body according to claim 2, characterized in that: The mouse lymphocytes are taken from a mouse with at least one marker of Foxp3 fluorescently stained.
4. The method for separating and purifying aTreg cells in mice according to claim 3, characterized in that: The CD4, the CD44 and the CD62L are stained by adding surface staining antibodies to the mouse lymphocytes.
5. The method for separating and purifying aTreg cells in mice according to claim 4, characterized in that: The surface staining antibodies for staining the CD4 include one or more of CD4-BV421, CD4-FITC, CD4-APC.
6. The method for separating and purifying aTreg cells in a mouse body according to claim 4, characterized in that: The surface staining antibodies for staining the CD44 include one or more of CD44-PE-cy7, CD44-PE, CD44-APC.
7. The method for separating and purifying aTreg cells in mice according to claim 4, wherein: The surface staining antibodies for staining the CD62L include one or more of CD62L-PC5.5, CD62L-Vioblue, CD62L-FITC.
8. The method for separating and purifying aTreg cells in mice according to claim 3, characterized in that: The Foxp3 of the mouse is fluorescently labeled with one or both of YFP and GFP.
9. The method for separating and purifying aTreg cells in a mouse body according to claim 1, wherein: The mouse lymphocytes are obtained by the following steps: taking mouse spleen and lymph node tissues, grinding them and filtering them through a 40μm filter to obtain a single cell suspension, adding erythrocyte lysate for 35 - 45s, then adding PBS buffer to stop the lysis, centrifuging at a speed of 1250 - 1350r / min for 5 minutes, discarding the supernatant, and washing twice with PBS buffer to obtain mouse lymphocytes.
10. The method for separating and purifying aTreg cells in a mouse body according to claim 1, characterized in that: The purity of the isolated and purified aTreg cells reaches 95% - 99%.