CN116858754A - Method for evaluating mouse sperm antigen specific T cell response - Google Patents
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Abstract
The application discloses a method for evaluating a mouse sperm antigen specific T cell response, and relates to the technical field of antigen specific T cells. The method for evaluating the mouse sperm antigen specific T cell response constructs a simple scheme based on flow cytometry by constructing a transgenic mouse with sperm specific expression of Ovalbumin (OVA) antigen and applying cell adoptive transfer, and is used for quantitatively analyzing antigen specific CD4+ T cell in vivo in the sperm antigen draining lymph node and spleen of the OVA immunized mouse, and the scheme quantifies the differentiation of the adoptive transfer OVA specific TCR transgenic CD4T (OT-2) cells, and can also be applied to the combination between Dendritic Cell (DC) subgroups and other types of cell interactions between T cells and other Antigen Presenting Cell (APC) subgroups.
Description
Technical Field
The application relates to the technical field of antigen-specific T cells, in particular to a method for evaluating a mouse sperm antigen-specific T cell response.
Background
Antigen Presenting Cells (APCs) refer to cells that can ingest and process antigens within cells and present antigen information to T lymphocytes. The antigen presenting cells generally referred to include cells expressing MHC class II molecules such as Dendritic Cells (DC), macrophages (Mphi), B lymphocytes, etc., and are also referred to as professional Antigen Presenting Cells (APCs). Dendritic Cells (DCs) are currently known to be the most potent APCs for antigen presentation, and are characterized by their ability to stimulate the activation and proliferation of naive T cells, whereas mfi, B lymphocytes, etc. are only able to stimulate activated T cells or memory T cells, and thus, DCs are the contributors to specific immune responses. With the intensive research into DC of different tissue origin, presently known DC sub-populations include classical DC (DCs) present in lymphoid tissues, blood and non-lymphoid tissues, and plasmacytoid DC (pDC) secreting type I interferon, respectively, with different phenotypes and functions. The main function of classical DCs is, among others, to induce specific immune responses against invading antigens and to maintain self-tolerance, while the main function of pdcs is to produce large amounts of type I interferons and to activate the corresponding T cells against microbial, in particular viral, infections. Binding between T cells and Antigen Presenting Cells (APCs) is essential for activating antigen specific T cells, but quantifying sperm antigen dependent T cell-APC binding is difficult.
Therefore, how to design a method for evaluating the antigen-specific T cell response of mouse sperm is a technical problem to be solved in the industry.
Disclosure of Invention
The application aims to provide a method for evaluating a mouse sperm antigen specific T cell response, and aims to solve the problem of quantitative sperm antigen dependent T cell-APC binding.
The application is realized in that the method for evaluating the mouse sperm antigen specific T cell response comprises the following steps: s1, separating, enriching and adoptively transferring CD4+TOT-2 cells into a wild 45.1 mouse body immunized by OVA sperms and an adjuvant; s2, collecting samples, performing enzyme digestion and staining cells of spleen lymph node tissues of the mice; s3, performing on-machine analysis on the sample after cell staining based on flow cytometry, and recording detected data; s4, analyzing the recorded data result.
Further, the step S1 includes the steps of: s11, constructing a CD4+ T cell serving as a carrier, and constructing a transgenic mouse for specifically expressing the heterologous chicken ovalbumin antigen on sperms; s12, performing adjuvant immunization operation on the CD45.1 mice.
Further, the step S11 includes the steps of: s111, designing and constructing a transgenic vector according to a design scheme, and verifying the correctness of a vector sequence through enzyme digestion and sequencing; s112, microinjection is carried out on a transgenic carrier sample, the transgenic carrier sample is microinjected into fertilized eggs of a mouse with a C57BL/6JGpt background, and then the fertilized eggs which survive after injection are transplanted into a pseudopregnant female mouse, and the pregnant female mouse is born; s113, carrying out gene identification on an F0 generation mouse, cutting the tail and the toe numbers of F0 generation mice born by a receptor mouse in 5-7 days, extracting genome DNA, carrying out PCR identification, and confirming the genotype; s114, recording and analyzing the results to ensure that mice with sperm specifically expressing the OVA antigen are successfully constructed.
Further, the step S12 includes the steps of: s121, mice expressing OVA specifically were euthanized one day prior to OT-2 cell transfection, and sperm cell suspensions expressing OVA and FCA were used in accordance with 1:1, uniformly mixing and emulsifying, and immunizing a right hind foot pad of a homologous CD45.1 mouse, wherein the injection amount of each foot pad is 20uL, and the operation time is 30 seconds/mouse; s122, isolating CD4+ T cells from adult male OVA specific T cell receptor transgenic OT-2 mice, wherein a CD45.1 mouse with homology is used as a receptor; s123, 5X10 of CD45.2 donor OT-2 cells under mild isoflurane anesthesia 6 The individual cells were transferred back to wild-type CD45.1 receptor mice immunized with OVA and adjuvant 1 day ago.
Further, the step S122 includes the steps of: s1221, euthanizing donor OT-2 mice and collecting spleen and all subcutaneous lymph nodes in a 100um cell filter placed with 2mL pre-chilledIn a 5cm culture dish of the culture medium, the precooling culture medium is RPMI1640 culture medium which contains 10% FBS; i. minced tissue fragments were ground with a sterile syringe plunger of a 3mL syringe at the bottom of a 100um cell filter; ii. 2mL of RPMI1640 digestion medium containing 5mg/mL collagenase D was added, and 10% FBS was added to form a final concentration of 2.5mg/mL collagenase D; iii, placing the petri dish in 5% CO 2 In an incubator and incubated at 37℃for 30 minutes; s1222, cutting spleen and all lymph nodes into pieces with the size of 1mm by using fine scissors; s1223, sorting CD4T cells by using a sorting kit according to the instruction; s1224 washing and suspending the isolated OT-2 cells in ice-cold phosphate buffer at a concentration of 1X10 7 cells/mL。
Further, 1X10 is obtained from one OT-2 mouse in the step S122 8 Total spleen and lymph node cells, 1.5-2.0x10 7 Individual cells were isolated to cd4+ot-2 cells with a purity of 90% -95% by using a kit.
Further, the step S2 includes the steps of: s21, collecting a sample of lymph node cell tissues of the mice, and performing enzyme digestion on the sample; s22, staining the cell tissues after enzyme digestion, and staining the cell surface markers by using an antibody.
Further, the step S21 includes the steps of: s211, before euthanizing the mice, by placing 1x1cm at the bottom of each well of a 24-well plate 2 Is 100um nylon mesh; a. 200uL of medium, RPMI1640 medium containing 10% FBS, was added to each well; b. preparing as many wells as necessary so that one mouse subcutaneous lymph node per well, and placing the plates on ice when collected; s212, euthanizing the mice 7 days after OT-2 cell transfer, and collecting inguinal region draining lymph nodes; s213, mechanically destroying subcutaneous lymph nodes; a. cutting subcutaneous lymph nodes into small pieces with fine scissors; b. subcutaneous lymphatic agglomerates were ground on nylon mesh with a plunger of a 1mL syringe, gently stirred; c. the scissors and plunger were washed with 800uLRPMI1640 and 10% FBS to a volume of 1ml in the well; s214, digestion of subcutaneous lymph nodes, addition of 10uL of 0.25g/mL collagenase D per well, and 5% CO at 37 ℃C 2 Culturing in an incubator for 30 minutes; s215 gently pipetting up and down the medium in the wells with a 1mL micropipette, collecting the digested subcutaneous lymph node cell suspension so that any residue of subcutaneous lymph node tissue will separate from the nylon mesh; s216, placing 1X1cm on top of 1.5mL tube 2 Nylon mesh and passing the whole digested cell suspension through the mesh; s217, taking 10uL for cell counting, washing with 0.6mL ice-cold phosphate buffer, passing the washing solution through a nylon net, and placing the cell suspension on ice; s218, centrifugation at 500xg4 degrees for 5 minutes, supernatant removed, and cell suspension transferred to 96-well V-bottom plate by gently pipetting cells resuspended in 200uL of RPMI1640 with 10% fbs; s219, centrifuging at 850xg for 2 minutes at 4 ℃, and then pouring the supernatant into a water tank or a waste container; s2110, cleaning cells; a. gently rotate the plate; b. washing cells by multichannel pipettor addition of 200 uL/well EDTA-free phosphate buffer; c. centrifuging at 450Xg for 5 minutes at 4 ℃; d. the supernatant was poured into a waste liquid container.
Further, the step S22 includes the steps of: s221, dying cells; a. the cell pellet was turned into a single cell suspension by gentle swirling and 20 uL/Kong Ranliao was added; b. gently rotating the plate to suspend the cells; c. incubating the plates on ice for 15-30 minutes; d. washing the cells; s222, staining a cell surface marker; a. adding 10 uL/hole Fc blocking solution; b. suspending cells by gentle vortexing; c. incubation on ice for 15 min; d. adding 10 uL/well of cell surface staining antibody mixture without washing; e. suspending the cells by gentle vortexing; f. placing the culture plate on ice for 15-30 minutes; g. the cells were washed.
Further, the step S3 includes the steps of: s31, adding 100 mu l of working solution in each reaction, fully suspending cells, incubating for 1h at the temperature of 4 ℃ in dark, preparing 1 XPermeabizationBuffer in the double-distilled water in situ, preparing an intracellular flow type staining antibody mixture by using the solution, wherein the reaction system comprises transcription factors and related cytokine antibodies, diluting the antibodies according to a ratio of 1:200, fully mixing the antibodies uniformly for later use; s32, adding 500 μl of 1 Xpermabilizationfuffer into the cell suspension with the fixed broken core in the step S31, centrifuging at 700 Xg and 4 ℃ for 5min, and removing the supernatant to obtain a cell precipitate; s33, re-suspending the cells by using 100 μl of the antibody mixture, fully mixing, and then incubating for 40min at 4deg.C in the dark; s34, after the reaction is finished, the cells are washed, finally, 200-300 mu l of flow staining buffer is used for resuspension, the cells are filtered by passing the cell suspension through a small piece of nylon net, and the cells are collected in a 5mL round bottom polystyrene tube.
Compared with the prior art, the method for evaluating the mouse sperm antigen specific T cell response has the following beneficial effects:
the application has the following beneficial effects: a simple flow cytometry-based protocol for quantitative analysis of antigen-specific cd4+ T cell responses in the sperm antigen draining lymph nodes and spleen of OVA immunized mice was constructed by constructing transgenic mice that express Ovalbumin (OVA) antigen specifically and applying cell adoptive transfer, which quantifies differentiation of adoptive-transferred OVA-specific TCR transgenic CD4T (OT-2) cells, which can also be applied to binding between Dendritic Cell (DC) subpopulations and other types of cell interactions between T cells and other Antigen Presenting Cell (APC) subpopulations.
Drawings
FIG. 1 is a flow chart of a method of evaluating a mouse sperm antigen specific T cell response in accordance with the present application;
FIG. 2 is a flow chart of a method of cell transfer and adjuvant immunization according to the present application;
FIG. 3 is a flow chart of a method of constructing a mouse with sperm-specific expression of OVA antigen in accordance with the present application;
FIG. 4 is a flow chart of a method of the application for adjuvant immunization of wild type CD45.1 mice;
FIG. 5 is a flow chart of a method of the present application for isolating CD4+ T cells in transgenic OT-2 mice;
FIG. 6 is a flow chart of a method of collecting and staining mouse lymph node cells according to the present application;
FIG. 7 is a flow chart of a method of collecting and enzymatically digesting a sample of mouse lymph node cells in accordance with the present application;
FIG. 8 is a flow chart of a method of staining cells according to the present application;
FIG. 9 is a flow chart of a method of performing on-machine analysis of stained cell samples according to the present application.
Detailed Description
The present application will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present application more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.
The implementation of the present application will be described in detail below with reference to specific embodiments.
The same or similar reference numerals in the drawings of the present embodiment correspond to the same or similar components; in the description of the present application, it should be understood that, if there is an azimuth or positional relationship indicated by terms such as "upper", "lower", "left", "right", etc., based on the azimuth or positional relationship shown in the drawings, it is only for convenience of describing the present application and simplifying the description, but it is not indicated or implied that the apparatus or element referred to must have a specific azimuth, be constructed and operated in a specific azimuth, and thus terms describing the positional relationship in the drawings are merely illustrative and should not be construed as limitations of the present patent, and specific meanings of the terms described above may be understood by those skilled in the art according to specific circumstances.
Referring to fig. 1-9, a preferred embodiment of the present application is provided.
A method of evaluating a mouse sperm antigen specific T cell response comprising the steps of: s1, separating, enriching and adoptively transferring CD4+TOT-2 cells into a wild 45.1 mouse body immunized by OVA sperms and an adjuvant; s2, collecting samples, performing enzyme digestion and staining cells of spleen lymph node tissues of the mice; s3, performing on-machine analysis on the sample after cell staining based on flow cytometry, and recording detected data; s4, analyzing the recorded data result.
Specifically, S1 includes the steps of: s11, constructing a CD4+ T cell serving as a carrier, and constructing a transgenic mouse for specifically expressing the heterologous chicken ovalbumin antigen on sperms; s12, performing adjuvant immunization operation on the CD45.1 mice.
In this embodiment, CD45.2OT-2 mice were prepared as well as CD45.1 mice, OT-2 mice had CD4+ T cell receptor specific recognition OVA323-339 peptide, OT-2 mice had CD4/CD8 peripheral blood T cell ratio increased 4-fold, lymph node T cells exhibited dose-dependent proliferative responses to specific ovalbumin ligands, allelic variants CD45.1 and CD45.2 of mouse CD45 were expressed in different mouse lineages, most mouse lineages expressed CD45.2, while CD45.1 was expressed in a few mouse lineages, CD45.1 and CD45.2 were often used to track the origin of mouse hematopoietic cells after bone marrow transplantation, a specific marker to distinguish lymphoma leukemia from non-hematopoietic tissue tumors, for the subsequent construction of transgenic mice that required to specifically express xenogeneic chicken ovalbumin antigen on sperm.
Specifically, S11 includes the steps of: s111, designing and constructing a transgenic vector according to a design scheme, and verifying the correctness of a vector sequence through enzyme digestion and sequencing; s112, microinjection is carried out on a transgenic carrier sample, the transgenic carrier sample is microinjected into fertilized eggs of a mouse with a C57BL/6JGpt background, and then the fertilized eggs which survive after injection are transplanted into a pseudopregnant female mouse, and the pregnant female mouse is born; s113, carrying out gene identification on an F0 generation mouse, cutting the tail and the toe numbers of F0 generation mice born by a receptor mouse in 5-7 days, extracting genome DNA, carrying out PCR identification, and confirming the genotype; s114, recording and analyzing the results to ensure that mice with sperm specifically expressing the OVA antigen are successfully constructed.
In this embodiment, the method is used to transplant the surviving fertilized ovum into the pseudopregnant female mouse by microinjection, so that the fertilized sperm specifically expresses OVA antigen mouse, PCR is a short term for polymerase chain reaction, is a method for synthesizing specific DNA fragments by in vitro enzymatic synthesis, and consists of reactions such as high temperature denaturation, low temperature annealing, temperature adaptation extension and the like for 1 period, and the method is circularly carried out, so that the target DNA can be rapidly amplified, and can be used for basic researches such as gene separation cloning, nucleic acid sequence analysis and the like, and diagnosis of diseases and any places with DNA and RNA.
Concrete embodimentsS12 includes the following steps: s121, mice expressing OVA specifically were euthanized one day prior to OT-2 cell transfection, and sperm cell suspensions expressing OVA and FCA were used in accordance with 1:1, uniformly mixing and emulsifying, and immunizing a right hind foot pad of a homologous CD45.1 mouse, wherein the injection amount of each foot pad is 20uL, and the operation time is 30 seconds/mouse; s122, isolating CD4+ T cells from adult male OVA specific T cell receptor transgenic OT-2 mice, wherein a CD45.1 mouse with homology is used as a receptor; s123, 5X10 of CD45.2 donor OT-2 cells under mild isoflurane anesthesia 6 The individual cells were transferred back to wild-type CD45.1 receptor mice immunized with OVA and adjuvant 1 day ago.
In this embodiment, FCA is freund's complete adjuvant in freund's adjuvant, which is generally regarded as the most commonly used immunological adjuvant in the current animal experiments, is commonly used for animal immunization to prepare antibodies, and can sustain sustained release of antigens, and simultaneously, nonspecifically enhance specific immune responses of organisms to the antigens, enhance immunogenicity of the corresponding antigens or change immune response types, and is essentially a mixture containing non-metabolic oil, surfactant or inactivated bacteria, and water-in-oil emulsion is formed by mixing water-soluble antigen solutions in equal volumes, the antigen emulsion not only increases the distribution range of antigens in vivo so as to enhance interactions with relevant cells, but also enables the antigens to be slowly released in organisms, prolongs antigen stimulation and enhances local immune responses, and the freund's adjuvant is divided into freund's complete adjuvant and freund's incomplete adjuvant; the homologous CD45.1 mouse background was B6.SJL-PtprcaPep3b/BoyJ.
Specifically, S122 includes the steps of: s1221, euthanizing donor OT-2 mice, and collecting spleen and all subcutaneous lymph nodes in a 100um cell filter placed in a 5cm dish with 2mL pre-chilled medium, RPMI1640 medium containing 10% FBS; i. minced tissue fragments were ground with a sterile syringe plunger of a 3mL syringe at the bottom of a 100um cell filter; ii. 2mL of RPMI1640 digestion medium containing 5mg/mL collagenase D was added, and 10% FBS was added to form a final concentration of 2.5mg/mL collagenase D; iii, placing the petri dish in 5% CO 2 In an incubator and incubated at 37℃for 30 minutes; s1222, cutting spleen and all lymph nodes into pieces with the size of 1mm by using fine scissors; s1223, sorting CD4T cells by using a sorting kit according to the instruction; s1224 washing and suspending the isolated OT-2 cells in ice-cold phosphate buffer at a concentration of 1X10 7 cells/mL。
In the embodiment, the FBS is fetal bovine serum, 10% of FBS is added in general cell culture to facilitate cell growth, and the fetal bovine serum is added according to the volume proportion, so that the fetal bovine serum is always the gold standard of a mammalian cell culture supplement and is widely applied to cell biology; the RPMI1640 cell culture medium is a classical medium and can be used for culturing lymphocytes and various cells.
Specifically, 1X10 was obtained from one OT-2 mouse in step S122 8 Total spleen and lymph node cells, 1.5-2.0x10 7 Individual cells were isolated to cd4+ot-2 cells with a purity of 90% -95% by using a kit.
In this embodiment, the kit used is a kit of BD or STEMCELL for isolating and culturing cells.
Specifically, S2 includes the steps of: s21, collecting a sample of lymph node cell tissues of the mice, and performing enzyme digestion on the sample; s22, staining the cell tissues after enzyme digestion, and staining the cell surface markers by using an antibody.
In this embodiment, the purpose of enzyme digestion is to expose the antigen, increase the permeability of cells and tissues, so as to facilitate the maximum binding of antibodies and antigens, and the subsequent on-machine analysis of samples can be performed by collecting, enzyme-digesting and staining the lymph node cell tissue of the mouse, and the operation time of step S21 is 1h.
Specifically, S21 includes the steps of: s211, before euthanizing the mice, by placing 1x1cm at the bottom of each well of a 24-well plate 2 Is 100um nylon mesh; a. 200uL of medium, RPMI1640 medium containing 10% FBS, was added to each well; b. as many wells as necessary are prepared so that one mouse subcutaneous lymph node per well will be collectedThe plates are placed on ice; s212, euthanizing the mice 7 days after OT-2 cell transfer, and collecting inguinal region draining lymph nodes; s213, mechanically destroying subcutaneous lymph nodes; a. cutting subcutaneous lymph nodes into small pieces with fine scissors; b. subcutaneous lymphatic agglomerates were ground on nylon mesh with a plunger of a 1mL syringe, gently stirred; c. the scissors and plunger were washed with 800uLRPMI1640 and 10% FBS to a volume of 1ml in the well; s214, digestion of subcutaneous lymph nodes, addition of 10uL of 0.25g/mL collagenase D per well, and 5% CO at 37 ℃C 2 Culturing in an incubator for 30 minutes; s215 gently pipetting up and down the medium in the wells with a 1mL micropipette, collecting the digested subcutaneous lymph node cell suspension so that any residue of subcutaneous lymph node tissue will separate from the nylon mesh; s216, placing 1X1cm on top of 1.5mL tube 2 Nylon mesh and passing the whole digested cell suspension through the mesh; s217, taking 10uL for cell counting, washing with 0.6mL ice-cold phosphate buffer, passing the washing solution through a nylon net, and placing the cell suspension on ice; s218, centrifugation at 500xg4 degrees for 5 minutes, supernatant removed, and cell suspension transferred to 96-well V-bottom plate by gently pipetting cells resuspended in 200uL of RPMI1640 with 10% fbs; s219, centrifuging at 850xg for 2 minutes at 4 ℃, and then pouring the supernatant into a water tank or a waste container; s2110, cleaning cells; a. gently rotate the plate; b. washing cells by multichannel pipettor addition of 200 uL/well EDTA-free phosphate buffer; c. centrifuging at 450Xg for 5 minutes at 4 ℃; d. the supernatant was poured into a waste liquid container.
In this embodiment, 850xg represents the magnitude of centrifugal force, g is a multiple of gravitational acceleration, and x represents the magnitude of g value, so that the rotational speed of the centrifuge can be calculated according to the formula g=1.118x10-5 xrxn 2; the phosphate buffer solution consists of a mixed solution of sodium dihydrogen phosphate and sodium dihydrogen phosphate, wherein the sodium dihydrogen phosphate is alkaline, and the sodium dihydrogen phosphate is acidic; EDTA is one of the digestive juice, and the digestive juice of tissue cells is used for the digestion and dispersion of the adherent growth cells in the in-vitro culture of tissue cells, the dispersion of tissue cells in primary cell culture and the culture of passage cells. Its function is mainly to hydrolyze intercellular proteins, disperse tissue or adherent cells into single cells, and make cell suspension for further experiments.
Specifically, S22 includes the steps of: s221, dying cells; a. the cell pellet was turned into a single cell suspension by gentle swirling and 20 uL/Kong Ranliao was added; b. gently rotating the plate to suspend the cells; c. incubating the plates on ice for 15-30 minutes; d. washing the cells; s222, staining a cell surface marker; a. adding 10 uL/hole Fc blocking solution; b. suspending cells by gentle vortexing; c. incubation on ice for 15 min; d. adding 10 uL/well of cell surface staining antibody mixture without washing; e. suspending the cells by gentle vortexing; f. placing the culture plate on ice for 15-30 minutes; g. the cells were washed.
In this embodiment, the dye is ZombieUV or Zombieaqua, which is diluted 1:1000 in hydrochloric acid buffer solution, and ZombieUV and Zombieaqua Zombie are both Zombie ultraviolet light, which is amine reactive fluorescent dye, impermeable to living cells but permeable to cells with damaged membranes, thus it can be used to evaluate the survival and death states of mammalian cells, zombieUV is a polar water-soluble dye providing purple fluorescence, making it suitable for multicolor detection, the principle of Zombie dye can combine with free amine on the cell surface and in the membrane to give out high intensity fluorescent signals due to the change of cell membrane permeability, zombieaqua is a polar water-soluble dye providing very bright green fluorescence, making it suitable for multicolor detection; wherein the operation of washing the cells is the same as that in step S2110.
Specifically, S3 includes the steps of: s31, adding 100 mu l of working solution in each reaction, fully suspending cells, incubating for 1h at the temperature of 4 ℃ in dark, preparing 1 XPermeabizationBuffer in the double-distilled water in situ, preparing an intracellular flow type staining antibody mixture by using the solution, wherein the reaction system comprises transcription factors and related cytokine antibodies, diluting the antibodies according to a ratio of 1:200, fully mixing the antibodies uniformly for later use; s32, adding 500 μl of 1 Xpermabilizationfuffer into the cell suspension with the fixed broken core in the step S31, centrifuging at 700 Xg and 4 ℃ for 5min, and removing the supernatant to obtain a cell precipitate; s33, re-suspending the cells by using 100 μl of the antibody mixture, fully mixing, and then incubating for 40min at 4deg.C in the dark; s34, after the reaction is finished, the cells are washed, finally, 200-300 mu l of flow staining buffer is used for resuspension, the cells are filtered by passing the cell suspension through a small piece of nylon net, and the cells are collected in a 5mL round bottom polystyrene tube.
In this embodiment, the working solution is Foxp3 fix/permeination working solution (diluted with double distilled water, 10 times diluted, 10×permeination buffer, now prepared for use), foxp3 fix/permeination working solution is the english name of Foxp3 fix/permeabilize buffer, permeination buffer is the english name of permeabilize buffer, after diluting it 10 times, it is the english name of working solution used later, the fixed rupture time is no more than 18h at most in the incubation process, the operation time of step S3 and step S22 is 2h, in this step, the operation of cell cleaning is the same as in step S2110.
The technical scheme provided by the embodiment of the application at least has the following technical effects or advantages:
a simple flow cytometry-based protocol for quantitative analysis of antigen-specific cd4+ T cell in vivo responses in the sperm antigen draining lymph nodes and spleen of OVA-immunized mice was constructed by constructing transgenic mice that express Ovalbumin (OVA) antigen specifically and applying cell adoptive transfer, which quantifies differentiation of adoptive-transferred OVA-specific TCR transgenic CD4T (OT-2) cells, which can also be applied to binding between Dendritic Cell (DC) subpopulations and other types of cell interactions between T cells and other Antigen Presenting Cell (APC) subpopulations; since this protocol is intended to detect cell differentiation between cell adoptive transfer donor OT-2 cells and sperm antigens in skin draining lymph nodes, the cell preparation procedure is optimized for OT-2CD4T cells transferred in lymph node spleen, and this flow cytometry-based method, while efficient for quantitative analysis of OT-2 cell differentiation, may require other imaging-based methods such as immunohistochemistry and imaging flow cytometry to fully validate and interpret the data. For example, immunocytochemistry analysis in combination with this protocol may provide a better presentation of OT-2 activation. Furthermore, this approach can provide a powerful tool for analyzing the molecular characteristics of antigen-specific T cell interacting cells under specific conditions in combination with single cell RNA sequencing.
The foregoing description of the preferred embodiments of the application is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the application.
Claims (10)
1. A method of evaluating a mouse sperm antigen specific T cell response comprising the steps of:
s1, separating, enriching and adoptively transferring CD4+TOT-2 cells into a wild 45.1 mouse body immunized by OVA sperms and an adjuvant;
s2, collecting samples, performing enzyme digestion and staining cells of spleen lymph node tissues of the mice;
s3, performing on-machine analysis on the sample after cell staining based on flow cytometry, and recording detected data;
s4, analyzing the recorded data result.
2. The method of evaluating a mouse sperm antigen specific T cell response as described in claim 1, wherein said S1 comprises the steps of:
s11, constructing a CD4+ T cell serving as a carrier, and constructing a transgenic mouse for specifically expressing the heterologous chicken ovalbumin antigen on sperms;
s12, performing adjuvant immunization operation on the CD45.1 mice.
3. The method of evaluating a mouse sperm antigen specific T cell response as described in claim 2, wherein said S11 comprises the steps of:
s111, designing and constructing a transgenic vector according to a design scheme, and verifying the correctness of a vector sequence through enzyme digestion and sequencing;
s112, microinjection is carried out on a transgenic carrier sample, the transgenic carrier sample is microinjected into fertilized eggs of a mouse with a C57BL/6JGpt background, and then the fertilized eggs which survive after injection are transplanted into a pseudopregnant female mouse, and the pregnant female mouse is born;
s113, carrying out gene identification on an F0 generation mouse, cutting the tail and the toe numbers of F0 generation mice born by a receptor mouse in 5-7 days, extracting genome DNA, carrying out PCR identification, and confirming the genotype;
s114, recording and analyzing the results to ensure that mice with sperm specifically expressing the OVA antigen are successfully constructed.
4. The method of evaluating a mouse sperm antigen specific T cell response as described in claim 2, wherein said S12 comprises the steps of:
s121, mice expressing OVA specifically were euthanized one day prior to OT-2 cell transfection, and sperm cell suspensions expressing OVA and FCA were used in accordance with 1:1, uniformly mixing and emulsifying, and immunizing a right hind foot pad of a homologous CD45.1 mouse, wherein the injection amount of each foot pad is 20uL, and the operation time is 30 seconds/mouse;
s122, isolating CD4+ T cells from adult male OVA specific T cell receptor transgenic OT-2 mice, wherein a CD45.1 mouse with homology is used as a receptor;
s123, 5X10 of CD45.2 donor OT-2 cells under mild isoflurane anesthesia 6 The individual cells were transferred back to wild-type CD45.1 receptor mice immunized with OVA and adjuvant 1 day ago.
5. The method of evaluating a mouse sperm antigen specific T cell response as described in claim 4, wherein said S122 comprises the steps of:
s1221, euthanizing donor OT-2 mice, and collecting spleen and all subcutaneous lymph nodes in a 100um cell filter placed in a 5cm dish with 2mL of pre-chilled medium, RPMI1640 medium containing 10% FBS;
i. minced tissue fragments were ground with a sterile syringe plunger of a 3mL syringe at the bottom of a 100um cell filter;
ii. 2mL of RPMI1640 digestion medium containing 5mg/mL collagenase D was added, and 10% FBS was added to form a final concentration of 2.5mg/mL collagenase D;
iii, placing the petri dish in 5% CO 2 In an incubator and incubated at 37℃for 30 minutes;
s1222, cutting spleen and all lymph nodes into pieces with the size of 1mm by using fine scissors;
s1223, sorting CD4T cells by using a sorting kit according to the instruction;
s1224 washing and suspending the isolated OT-2 cells in ice-cold phosphate buffer at a concentration of 1X10 7 cells/mL。
6. The method of evaluating a mouse sperm antigen specific T cell response as described in claim 5, wherein: 1X10 was obtained from one OT-2 mouse in the S122 step 8 Total spleen and lymph node cells, 1.5-2.0x10 7 Individual cells were isolated to cd4+ot-2 cells with a purity of 90% -95% by using a kit.
7. The method of evaluating a mouse sperm antigen specific T cell response as described in claim 1, wherein said S2 comprises the steps of:
s21, collecting a sample of lymph node cell tissues of the mice, and performing enzyme digestion on the sample;
s22, staining the cell tissues after enzyme digestion, and staining the cell surface markers by using an antibody.
8. The method of evaluating a mouse sperm antigen specific T cell response as described in claim 7, wherein said S21 comprises the steps of:
s211, before euthanizing the mice, by placing 1x1cm at the bottom of each well of a 24-well plate 2 Is 100um nylon mesh;
a. 200uL of medium, RPMI1640 medium containing 10% FBS, was added to each well;
b. preparing as many wells as necessary so that one mouse subcutaneous lymph node per well, and placing the plates on ice when collected;
s212, euthanizing the mice 7 days after OT-2 cell transfer, and collecting inguinal region draining lymph nodes;
s213, mechanically destroying subcutaneous lymph nodes;
a. cutting subcutaneous lymph nodes into small pieces with fine scissors;
b. subcutaneous lymphatic agglomerates were ground on nylon mesh with a plunger of a 1mL syringe, gently stirred;
c. the scissors and plunger were washed with 800uLRPMI1640 and 10% FBS to a volume of 1ml in the well;
s214, digestion of subcutaneous lymph nodes, addition of 10uL of 0.25g/mL collagenase D per well, and 5% CO at 37 ℃C 2 Culturing in an incubator for 30 minutes;
s215 gently pipetting up and down the medium in the wells with a 1mL micropipette, collecting the digested subcutaneous lymph node cell suspension so that any residue of subcutaneous lymph node tissue will separate from the nylon mesh;
s216, placing 1X1cm on top of 1.5mL tube 2 Nylon mesh and passing the whole digested cell suspension through the mesh;
s217, taking 10uL for cell counting, washing with 0.6mL ice-cold phosphate buffer, passing the washing solution through a nylon net, and placing the cell suspension on ice;
s218, centrifugation at 500xg4 degrees for 5 minutes, supernatant removed, and cell suspension transferred to 96-well V-bottom plate by gently pipetting cells resuspended in 200uL of RPMI1640 with 10% fbs;
s219, centrifuging at 850xg for 2 minutes at 4 ℃, and then pouring the supernatant into a water tank or a waste container;
s2110, cleaning cells;
a. gently rotate the plate;
b. washing cells by multichannel pipettor addition of 200 uL/well EDTA-free phosphate buffer;
c. centrifuging at 450Xg for 5 minutes at 4 ℃;
d. the supernatant was poured into a waste liquid container.
9. The method of evaluating a mouse sperm antigen specific T cell response as described in claim 7, wherein said S22 comprises the steps of:
s221, dying cells;
a. the cell pellet was turned into a single cell suspension by gentle swirling and 20 uL/Kong Ranliao was added;
b. gently rotating the plate to suspend the cells;
c. incubating the plates on ice for 15-30 minutes;
d. washing the cells;
s222, staining a cell surface marker;
a. adding 10 uL/hole Fc blocking solution;
b. suspending cells by gentle vortexing;
c. incubation on ice for 15 min;
d. adding 10 uL/well of cell surface staining antibody mixture without washing;
e. suspending the cells by gentle vortexing;
f. placing the culture plate on ice for 15-30 minutes;
g. the cells were washed.
10. The method of evaluating a mouse sperm antigen specific T cell response as described in claim 1, wherein said S3 comprises the steps of:
s31, adding 100 mu l of working solution in each reaction, fully suspending cells, incubating for 1h at the temperature of 4 ℃ in dark, preparing 1 XPermeabizationBuffer in the double-distilled water in situ, preparing an intracellular flow type staining antibody mixture by using the solution, wherein the reaction system comprises transcription factors and related cytokine antibodies, diluting the antibodies according to a ratio of 1:200, fully mixing the antibodies uniformly for later use;
s32, adding 500 mu l of 1× permeabilization buffer to the cell suspension with the fixed broken cores in the step S31, centrifuging at 700×g and 4 ℃ for 5min, and removing the supernatant to obtain a cell precipitate;
s33, re-suspending the cells by using 100 μl of the antibody mixture, fully mixing, and then incubating for 40min at 4deg.C in the dark;
s34, after the reaction is finished, the cells are washed, finally, 200-300 mu l of flow staining buffer is used for resuspension, the cells are filtered by passing the cell suspension through a small piece of nylon net, and the cells are collected in a 5mL round bottom polystyrene tube.
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