Detection reagent for immune cells and preparation and detection methods thereof

Abstract

The invention belongs to the technical field of immune cell detection, and particularly relates to a detection reagent for immune cells and a preparation and detection method of the detection reagent. S2, carrying out enzymolysis digestion; s3, preparing a single-cell suspension; s4, sorting the immunomagnetic beads; s5, carrying out flow dyeing; s6, assembling the kit; according to the immune cell detection reagent and the preparation and detection method, through the synergistic effect of different enzymes, tissues can be decomposed mildly and effectively, the excessive digestion situation possibly caused by only using a certain enzyme is prevented, damage to cell activity is reduced, the activity requirement of cells in subsequent detection is guaranteed, and then the accuracy of a detection result is guaranteed; secondly, various enzymes are used for decomposing different components in the tissue, so that the tissue can be digested into a single-cell suspension more quickly and thoroughly compared with a single enzyme, the cell yield is increased, and enough cells are provided for the subsequent steps of immunomagnetic bead sorting and the like.

Description

Technical Field

[0001] This invention belongs to the field of immune cell detection technology, specifically to the detection reagents, preparation and detection methods for immune cells. Background Technology

[0002] In the field of immune cell research and application, accurately obtaining specific types of immune cells is a key prerequisite for conducting subsequent experiments and research.

[0003] Furthermore, in immune cell detection, cell viability is a key indicator. Traditional methods that use only one enzyme are prone to over-digesting tissues and damaging cell viability. This can easily lead to low cell viability due to over-digestion, affecting the accuracy of the test results. At the same time, single-enzyme digestion cannot quickly digest tissues into single-cell suspensions, resulting in low cell yield.

[0004] Therefore, the present invention provides a detection reagent for immune cells and a method for its preparation and detection. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, at least one technical problem raised in the background art is solved.

[0006] The technical solution adopted by this invention to solve its technical problem is: the preparation method of the immune cell detection reagent of this invention includes the following steps: S1. Tissue pretreatment: Fresh tissue is rinsed three times in pre-cooled sterile phosphate buffer to remove blood and necrotic parts, and then cut into 1-2 mm³ pieces with sterile ophthalmic scissors. Add pre-cooled phosphate buffer, let stand for 5 minutes, and aspirate the floating fat and blood vessel fragments on the surface. S2. Enzymatic digestion: A mixture of 1-2 mg / mL collagenase IV and 20 μg / mL deoxyribonuclease I was used. 0.1 mg / mL hyaluronidase was added to dense tissue. Hank's balanced salt solution containing Ca²⁺ / Mg²⁺ was used as a buffer. The mixture was digested in a shaker at 37°C for 30-90 minutes, with gentle pipetting every 10 minutes until the tissue block became flocculent. The reaction was then stopped, and an equal volume of DuPont modified Eagle medium containing 10% fetal bovine serum was added to terminate the enzyme activity. S3. Preparation of single-cell suspension: The cells were filtered sequentially through 100μm and 40μm cell sieves, rinsed with phosphate buffer three times, centrifuged at 300×g for 5 minutes, and the cell pellet was collected at 4℃. Cell viability was measured to be >90%. S4. Immunomagnetic bead sorting: Differentiate clusters of 4 microbeads per 10 μL of 10 μL. 7The proportion of cells was determined by incubation in phosphate buffer containing 0.5% bovine serum albumin and 2 mM ethylenediaminetetraacetic acid, followed by separation by a cell sorting column at a flow rate of 1 mL / min. During washing, the sorting column was kept moist to avoid air bubbles. Positive cells were immediately resuspended in pre-cooled complete culture medium after elution. S5. Flow cytometry staining: The sorted immune cells are further analyzed and identified through live / dead staining, surface antibody staining, and resuspending of the cells to be tested. S6. Reagent kit assembly: Includes the special reagents and auxiliary materials from S1 to S5 above, and an instruction manual.

[0007] Furthermore, step S1 is performed entirely on ice. If the tissue in step S1 contains a significant amount of blood, it is treated with erythrocyte lysis buffer for 5 minutes before washing. The erythrocyte lysis buffer is prepared as follows: Weigh 8.02g NH4Cl, 1.0g KHCO3, and 0.037g ethylenediaminetetraacetic acid-Na2, dissolve them in deionized water, bring the volume to 1L, adjust the pH to 7.2-7.4, filter to sterilize, and store at room temperature.

[0008] Furthermore, the preparation method of the pre-cooled sterile phosphate buffer is as follows: Dissolve phosphate buffer powder in deionized water, adjust the pH to 7.2-7.4, filter to sterilize, and then pre-cool at 4°C for later use.

[0009] Furthermore, the preparation method of the Duchenne modified Eagle medium containing 10% fetal bovine serum is as follows: Add 10% v / v fetal bovine serum to the basic Duchenne modified Eagle medium, mix well, filter to sterilize, and store in a 4°C refrigerator.

[0010] Furthermore, the preparation method of the fetal bovine serum buffer containing 0.5% bovine serum albumin + 2mM ethylenediaminetetraacetic acid is as follows: Bovine serum albumin was dissolved in fetal bovine serum to achieve a bovine serum albumin concentration of 0.5% w / v. Then, ethylenediaminetetraacetic acid (EDTA) was added to achieve an EDTA concentration of 2 mM. After filtration and sterilization, the solution was stored at 4°C.

[0011] Furthermore, the preparation method of the pre-cooled complete culture medium is as follows: Based on the growth requirements of the target immune cells, DuPont modified Eagle medium was selected, 10% to 20% v / v fetal bovine serum was added, and growth factors and antibiotics were added in proportion. After mixing evenly, the mixture was filtered to remove bacteria and placed in a 4°C refrigerator for pre-cooling before use. The mass ratio of collagenase IV to deoxyribonuclease I is 1:0.01–1:0.05; The differentiation cluster 4 microbeads are magnetic nanoparticles conjugated with anti-differentiation cluster 4 antibodies, with a particle size of 50-200 nm.

[0012] Furthermore, take an appropriate amount of the sorted cell suspension, select the live or dead dye according to the experimental requirements, dilute it appropriately according to the operation manual before use, add an appropriate amount of live or dead dye, vortex mix, protect from light, and incubate at 4°C for 30 minutes. Surface antibody staining procedure: Add fluorescently labeled antibodies against the target immune cell surface markers, vortex to mix, incubate at 4°C in the dark for 30 minutes, and wash the cells twice with flow cytometry buffer to remove unbound antibodies. Resuspension for inspection: Resuspend the cells in flow cytometry buffer, filter them before flow cytometry analysis, and prepare them for flow cytometry analysis.

[0013] Furthermore, before assembling the kit, each reagent and auxiliary material undergoes quality testing, including testing for reagent purity, activity, and sterility, and testing for the integrity and sealing of auxiliary materials. The kit assembly includes: pre-cooled sterile phosphate buffer, enzymes, cell sieves, centrifuge tubes, magnetic beads, sorting columns, pre-cooled complete culture medium, flow cytometry-related antibodies, live / dead dyes, fixation / permeabilization kit, permeabilization solution, flow cytometry buffer, and an experimental operation manual. These components are assembled into the kit according to a reasonable layout and sequence. The operation manual includes the kit composition, usage methods, precautions, and result analysis.

[0014] The detection method for the detection reagent of immune cells, and the preparation method of the detection reagent of immune cells, include the following steps: S1. Sample preparation: A suspension of differentiated cluster 4+ T cells was obtained after tissue pretreatment, enzymatic digestion, single-cell suspension preparation and immunomagnetic bead sorting. S2, Flow cytometry staining: S201. Live and dead staining: Take an appropriate amount of sorted cell suspension, select live and dead stain according to experimental requirements, dilute appropriately according to the operation manual before use, add an appropriate amount of live and dead stain, vortex mix, protect from light, and incubate at 4°C for 30 minutes. S202, Surface antibody staining: Add fluorescently labeled antibodies against the surface markers of the target immune cells, vortex to mix, incubate at 4°C in the dark for 30 minutes, wash the cells twice with flow cytometry buffer to remove unbound antibodies; S203, Resuspending cells for testing: Resuspend cells in flow cytometry buffer and filter them with a cell sieve before running the flow cytometer to remove cell clumps and prevent clogging. S204. Flow cytometry detection: Add the filtered cell suspension to the sample tube of the flow cytometer, set the detection parameters, start the flow cytometer to detect, collect and analyze the data, and judge the activity of immune cells and the expression of surface markers based on the fluorescence signal. S3. Results Analysis: Based on the flow cytometry results, and following the results analysis section of the kit's instruction manual, determine the relevant characteristics of immune cells, such as the ratio of live to dead cells and the expression level of surface markers. If abnormal results occur, handle them according to the solutions provided in the operation manual.

[0015] The detection reagents for immune cells, and the preparation method of the detection reagents for immune cells, include tissue processing reagents, enzymatic digestion reagents, single-cell suspension preparation reagents, immunomagnetic bead sorting reagents, and flow cytometry detection reagents.

[0016] The beneficial effects of this invention are as follows: 1. In the S2 enzymatic digestion step, the synergistic action of different enzymes can more gently and effectively decompose tissues, preventing over-digestion that may occur when using only one enzyme, reducing damage to cell viability, ensuring the cell viability requirements for subsequent detection, and thus ensuring the accuracy of the detection results; secondly, multiple enzymes decompose different components in the tissue, which can digest the tissue into a single-cell suspension more quickly and thoroughly than a single enzyme, improving cell yield and providing a sufficient number of cells for subsequent steps such as immunomagnetic bead sorting; 2. In the S4 immunomagnetic bead sorting step, by reducing non-specific binding and preventing cell aggregation, target cells can bind to the magnetic beads more effectively and be sorted, significantly improving the target cell recovery rate. Secondly, by reducing non-specific binding of non-target cells to the magnetic beads and the contamination caused by cell aggregation, the purity of the sorted cells is higher, providing more accurate results for subsequent flow cytometry staining and detection. At the same time, the precisely designed sorting buffer provides a stable chemical environment for immunomagnetic bead sorting, reducing the fluctuation of sorting results caused by the instability of buffer composition, making the entire preparation method more reproducible and reliable. Attached Figure Description

[0017] The invention will now be further described with reference to the accompanying drawings.

[0018] Figure 1 This is a flowchart of the preparation process of the present invention. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Please see Figure 1 This embodiment provides a method for preparing a detection reagent for immune cells, including the following steps: S1. Tissue pretreatment: Fresh tissue is rinsed three times in pre-cooled sterile phosphate buffer to remove blood and necrotic parts, and then cut into 1-2 mm³ pieces with sterile ophthalmic scissors. Step S1 should be performed entirely on ice. If the tissue contains a significant amount of blood during Step S1, treat it with erythrocyte lysis buffer for 5 minutes before washing. The preparation method for erythrocyte lysis buffer is as follows: Weigh 8.02g NH4Cl, 1.0g KHCO3, and 0.037g ethylenediaminetetraacetic acid-Na2, dissolve them in deionized water, bring the volume to 1L, adjust the pH to 7.2-7.4, filter to sterilize, and store at room temperature.

[0021] The preparation method for pre-cooled sterile phosphate buffer is as follows: Dissolve phosphate buffer powder in deionized water, adjust the pH to 7.2-7.4, filter to sterilize, and then pre-cool at 4°C for later use.

[0022] Add pre-cooled phosphate buffer, let stand for 5 minutes, and aspirate the floating fat and blood vessel fragments on the surface. S2. Enzymatic digestion: A mixture of 1-2 mg / mL collagenase IV and 20 μg / mL deoxyribonuclease I was used. 0.1 mg / mL hyaluronidase was added to dense tissue. Hank's balanced salt solution containing Ca²⁺ / Mg²⁺ was used as a buffer. The mixture was digested in a shaker at 37°C for 30-90 minutes, with gentle pipetting every 10 minutes until the tissue block became flocculent. The reaction was then stopped, and an equal volume of DuPont modified Eagle medium containing 10% fetal bovine serum was added to terminate the enzyme activity. The mass ratio of collagenase IV to deoxyribonuclease I was 1:0.01–1:0.05; The preparation method for DuPont modified Eagle medium containing 10% fetal bovine serum is as follows: Add 10% v / v fetal bovine serum to the basic Duchenne modified Eagle medium, mix well, filter to sterilize, and store in a 4°C refrigerator. S3. Preparation of single-cell suspension: The cells were filtered sequentially through 100μm and 40μm cell sieves, rinsed with phosphate buffer three times, centrifuged at 300×g for 5 minutes, and the cell pellet was collected at 4℃. Cell viability was measured to be >90%. S4. Immunomagnetic bead sorting: Differentiate clusters of 4 microbeads per 10 μL of 10 μL. 7 The proportion of cells was determined by incubation in phosphate buffer containing 0.5% bovine serum albumin and 2 mM ethylenediaminetetraacetic acid, followed by separation by a cell sorting column at a flow rate of 1 mL / min. During washing, the sorting column was kept moist to avoid air bubbles. Positive cells were immediately resuspended in pre-cooled complete culture medium after elution. Differentiation cluster 4 microbeads are magnetic nanoparticles conjugated with anti-differentiation cluster 4 antibodies, with a particle size of 50-200 nm; The preparation method for fetal bovine serum buffer containing 0.5% bovine serum albumin + 2mM ethylenediaminetetraacetic acid is as follows: Bovine serum albumin was dissolved in fetal bovine serum to achieve a bovine serum albumin concentration of 0.5% w / v. Then, ethylenediaminetetraacetic acid (EDTA) was added to achieve an EDTA concentration of 2 mM. After filtration and sterilization, the solution was stored at 4°C.

[0023] The preparation method for pre-cooled complete culture medium is as follows: Based on the growth requirements of the target immune cells, DuPont modified Eagle medium was selected, 10% to 20% v / v fetal bovine serum was added, and growth factors and antibiotics were added in proportion. After mixing evenly, the mixture was filtered to remove bacteria and placed in a 4°C refrigerator for pre-cooling before use. S5. Flow cytometry staining: The sorted immune cells are further analyzed and identified through live / dead staining, surface antibody staining, and resuspending of the cells to be tested. S6. Reagent kit assembly: Includes the special reagents and auxiliary materials from S1 to S5 above, and an instruction manual.

[0024] Take an appropriate amount of the sorted cell suspension, select the live or dead dye according to the experimental requirements, dilute it appropriately according to the operation manual before use, add an appropriate amount of live or dead dye, vortex mix, and incubate at 4°C for 30 minutes in the dark. Surface antibody staining procedure: Add fluorescently labeled antibodies against the target immune cell surface markers, vortex to mix, incubate at 4°C in the dark for 30 minutes, and wash the cells twice with flow cytometry buffer to remove unbound antibodies. Resuspension for inspection: Resuspend the cells in flow cytometry buffer, filter them before flow cytometry analysis, and prepare them for flow cytometry analysis.

[0025] Before assembling the kit, each reagent and auxiliary material is subjected to quality testing, including testing for reagent purity, activity, and sterility, and testing for the integrity and sealing of auxiliary materials. The kit consists of: pre-cooled sterile phosphate buffer, enzymes, cell sieves, centrifuge tubes, magnetic beads, sorting columns, pre-cooled complete culture medium, flow cytometry-related antibodies, live / dead dyes, fixation / permeabilization kit, permeabilization solution, flow cytometry buffer, and an experimental operation manual. These components are assembled into the kit according to a reasonable layout and sequence. The operation manual includes the kit composition, usage methods, precautions, and result analysis.

[0026] The detection method for the detection reagent used for immune cells, and the preparation method for the detection reagent used for immune cells, include the following steps: S1. Sample preparation: A suspension of differentiated cluster 4+ T cells was obtained after tissue pretreatment, enzymatic digestion, single-cell suspension preparation and immunomagnetic bead sorting. S2, Flow cytometry staining: S201. Live and dead staining: Take an appropriate amount of sorted cell suspension, select live and dead stain according to experimental requirements, dilute appropriately according to the operation manual before use, add an appropriate amount of live and dead stain, vortex mix, protect from light, and incubate at 4°C for 30 minutes. S202, Surface antibody staining: Add fluorescently labeled antibodies against the surface markers of the target immune cells, vortex to mix, incubate at 4°C in the dark for 30 minutes, wash the cells twice with flow cytometry buffer to remove unbound antibodies; S203, Resuspending cells for testing: Resuspend cells in flow cytometry buffer and filter them with a cell sieve before running the flow cytometer to remove cell clumps and prevent clogging. S204. Flow cytometry detection: Add the filtered cell suspension to the sample tube of the flow cytometer, set the detection parameters, start the flow cytometer to detect, collect and analyze the data, and judge the activity of immune cells and the expression of surface markers based on the fluorescence signal. S3. Results Analysis: Based on the flow cytometry results, and following the results analysis section of the kit's instruction manual, determine the relevant characteristics of immune cells, such as the ratio of live to dead cells and the expression level of surface markers. If abnormal results occur, handle them according to the solutions provided in the operation manual.

[0027] Detection reagents for immune cells, and methods for preparing detection reagents for immune cells, including tissue processing reagents, enzymatic digestion reagents, single-cell suspension preparation reagents, immunomagnetic bead sorting reagents, and flow cytometry detection reagents.

[0028] Example 1 The preparation method of the detection reagent for immune cells includes the following steps: S1. Tissue pretreatment: Fresh tissue is rinsed three times in pre-cooled sterile phosphate buffer to remove blood and necrotic parts, and then cut into 2mm³ pieces with sterile ophthalmic scissors. Add pre-cooled phosphate buffer, let stand for 5 minutes, and aspirate the floating fat and blood vessel fragments on the surface. S2. Enzymatic digestion: A mixture of 1.5 mg / mL collagenase IV and 20 μg / mL deoxyribonuclease I was used. 0.1 mg / mL hyaluronidase was added to dense tissue. Hank's balanced salt solution containing Ca²⁺ / Mg²⁺ was used as a buffer. The mixture was digested in a shaker at 37°C for 30 minutes, with gentle pipetting every 10 minutes until the tissue block became flocculent. The reaction was then stopped, and an equal volume of DuPont modified Eagle medium containing 10% fetal bovine serum was added to terminate the enzyme activity. S3. Preparation of single-cell suspension: The cells were filtered sequentially through 100μm and 40μm cell sieves, rinsed with phosphate buffer three times, centrifuged at 300×g for 5 minutes, and the cell pellet was collected at 4℃. Cell viability was measured to be >90%. S4. Immunomagnetic bead sorting: Differentiate clusters of 4 microbeads per 10 μL of 10 μL. 7 The proportion of cells was determined by incubation in phosphate buffer containing 0.5% bovine serum albumin and 2 mM ethylenediaminetetraacetic acid, followed by separation by a cell sorting column at a flow rate of 1 mL / min. During washing, the sorting column was kept moist to avoid air bubbles. Positive cells were immediately resuspended in pre-cooled complete culture medium after elution. S5. Flow cytometry staining: The sorted immune cells are further analyzed and identified through live / dead staining, surface antibody staining, and resuspending of the cells to be tested. S6. Reagent kit assembly: Includes the special reagents and auxiliary materials from S1 to S5 above, and an instruction manual.

[0029] Example 2 The preparation method of the detection reagent for immune cells includes the following steps: S1. Tissue pretreatment: Fresh tissue is rinsed three times in pre-cooled sterile phosphate buffer to remove blood and necrotic parts, and then cut into 2mm³ pieces with sterile ophthalmic scissors. Add pre-cooled phosphate buffer, let stand for 5 minutes, and aspirate the floating fat and blood vessel fragments on the surface. S2. Enzymatic digestion: A mixture of 1.5 mg / mL collagenase IV and 20 μg / mL deoxyribonuclease I was used. 0.1 mg / mL hyaluronidase was added to dense tissue. Hank's balanced salt solution containing Ca²⁺ / Mg²⁺ was used as a buffer. The mixture was digested in a shaker at 37°C for 60 minutes, with gentle pipetting every 10 minutes until the tissue block became flocculent. The reaction was then stopped, and an equal volume of DuPont modified Eagle medium containing 10% fetal bovine serum was added to terminate the enzyme activity. S3. Preparation of single-cell suspension: The cells were filtered sequentially through 100μm and 40μm cell sieves, rinsed with phosphate buffer three times, centrifuged at 300×g for 5 minutes, and the cell pellet was collected at 4℃. Cell viability was measured to be >90%. S4. Immunomagnetic bead sorting: Differentiate clusters of 4 microbeads per 10 μL of 10 μL. 7 The proportion of cells was determined by incubation in phosphate buffer containing 0.5% bovine serum albumin and 2 mM ethylenediaminetetraacetic acid, followed by separation by a cell sorting column at a flow rate of 1 mL / min. During washing, the sorting column was kept moist to avoid air bubbles. Positive cells were immediately resuspended in pre-cooled complete culture medium after elution. S5. Flow cytometry staining: The sorted immune cells are further analyzed and identified through live / dead staining, surface antibody staining, and resuspending of the cells to be tested. S6. Reagent kit assembly: Includes the special reagents and auxiliary materials from S1 to S5 above, and an instruction manual.

[0030] Example 3 The preparation method of the detection reagent for immune cells includes the following steps: S1. Tissue pretreatment: Fresh tissue is rinsed three times in pre-cooled sterile phosphate buffer to remove blood and necrotic parts, and then cut into 2mm³ pieces with sterile ophthalmic scissors. Add pre-cooled phosphate buffer, let stand for 5 minutes, and aspirate the floating fat and blood vessel fragments on the surface. S2. Enzymatic digestion: A mixture of 1.5 mg / mL collagenase IV and 20 μg / mL deoxyribonuclease I was used. 0.1 mg / mL hyaluronidase was added to dense tissue. Hank's balanced salt solution containing Ca²⁺ / Mg²⁺ was used as the buffer. The mixture was digested in a shaker at 37°C for 90 minutes, with gentle pipetting every 10 minutes until the tissue block became flocculent. The reaction was then stopped, and an equal volume of DuPont modified Eagle medium containing 10% fetal bovine serum was added to terminate the enzyme activity. S3. Preparation of single-cell suspension: The cells were filtered sequentially through 100μm and 40μm cell sieves, rinsed with phosphate buffer three times, centrifuged at 300×g for 5 minutes, and the cell pellet was collected at 4℃. Cell viability was measured to be >90%. S4. Immunomagnetic bead sorting: Differentiate clusters of 4 microbeads per 10 μL of 10 μL. 7The proportion of cells was determined by incubation in phosphate buffer containing 0.5% bovine serum albumin and 2 mM ethylenediaminetetraacetic acid, followed by separation by a cell sorting column at a flow rate of 1 mL / min. During washing, the sorting column was kept moist to avoid air bubbles. Positive cells were immediately resuspended in pre-cooled complete culture medium after elution. S5. Flow cytometry staining: The sorted immune cells are further analyzed and identified through live / dead staining, surface antibody staining, and resuspending of the cells to be tested. S6. Reagent kit assembly: Includes the special reagents and auxiliary materials from S1 to S5 above, and an instruction manual.

[0031] The enzymatic hydrolysis times for Examples 1, 2, and 3 were 30, 60, and 90 minutes, respectively, with other conditions being the same (collagenase IV 1.5 mg / mL + DNase I 20 μg / mL, 37℃ shaker), as shown in Table 1. Table 1: Example Enzymatic hydrolysis time (min) Cell yield (×10 / g tissue) Cell viability (%) CD4+ T cell purity (%) Example 1 30 5.2± 0.38 92±2 85±3 Example 2 60 8.1 ±0.5 88±3 89±2 Example 3 90 8.3±0.4 75 ± 4 82 ± 4 Conclusion: As shown in Table 1, a 60-minute enzymatic hydrolysis time resulted in a yield of 8.1 × 10⁻⁶. 6 The optimal balance between (g / g) and activity (88%) was achieved, but extending the time to 90 minutes resulted in a significant decrease in activity.

[0032] Comparative Example 1 The preparation method of the detection reagent for immune cells includes the following steps: S1. Tissue pretreatment: Fresh tissue is rinsed three times in pre-cooled sterile phosphate buffer to remove blood and necrotic parts, and then cut into 1-2 mm³ pieces with sterile ophthalmic scissors. Add pre-cooled phosphate buffer, let stand for 5 minutes, and aspirate the floating fat and blood vessel fragments on the surface. S2. Enzymatic digestion: A mixture of 1 mg / mL collagenase IV and 20 μg / mL deoxyribonuclease I was used. 0.1 mg / mL hyaluronidase was added to dense tissue. Hank's balanced salt solution containing Ca²⁺ / Mg²⁺ was used as the buffer. The mixture was digested in a shaker at 37°C for 60 minutes, with gentle pipetting every 10 minutes until the tissue block became flocculent. The reaction was then stopped, and an equal volume of DuPont modified Eagle medium containing 10% fetal bovine serum was added to terminate the enzyme activity. S3. Preparation of single-cell suspension: The cells were filtered sequentially through 100μm and 40μm cell sieves, rinsed with phosphate buffer three times, centrifuged at 300×g for 5 minutes, and the cell pellet was collected at 4℃. Cell viability was measured to be >90%. S4. Immunomagnetic bead sorting: Differentiate clusters of 4 microbeads per 10 μL of 10 μL. 7The proportion of cells was determined by incubation in phosphate buffer containing 0.5% bovine serum albumin and 0 mM ethylenediaminetetraacetic acid, followed by separation by a cell sorting column at a flow rate of 1 mL / min. During washing, the sorting column was kept moist to avoid air bubbles. Positive cells were immediately resuspended in pre-cooled complete culture medium after elution. S5. Flow cytometry staining: The sorted immune cells are further analyzed and identified through live / dead staining, surface antibody staining, and resuspending of the cells to be tested. S6. Reagent kit assembly: Includes the special reagents and auxiliary materials from S1 to S5 above, and an instruction manual.

[0033] Comparative Example 2 The preparation method of the detection reagent for immune cells includes the following steps: S1. Tissue pretreatment: Fresh tissue is rinsed three times in pre-cooled sterile phosphate buffer to remove blood and necrotic parts, and then cut into 1-2 mm³ pieces with sterile ophthalmic scissors. Add pre-cooled phosphate buffer, let stand for 5 minutes, and aspirate the floating fat and blood vessel fragments on the surface. S2. Enzymatic digestion: A mixture of 1 mg / mL collagenase IV and 20 μg / mL deoxyribonuclease I was used. 0.1 mg / mL hyaluronidase was added to dense tissue. Hank's balanced salt solution containing Ca²⁺ / Mg²⁺ was used as the buffer. The mixture was digested in a shaker at 37°C for 60 minutes, with gentle pipetting every 10 minutes until the tissue block became flocculent. The reaction was then stopped, and an equal volume of DuPont modified Eagle medium containing 10% fetal bovine serum was added to terminate the enzyme activity. S3. Preparation of single-cell suspension: The cells were filtered sequentially through 100μm and 40μm cell sieves, rinsed with phosphate buffer three times, centrifuged at 300×g for 5 minutes, and the cell pellet was collected at 4℃. Cell viability was measured to be >90%. S4. Immunomagnetic bead sorting: Differentiate clusters of 4 microbeads per 10 μL of 10 μL. 7 The proportion of cells was determined by incubation in phosphate buffer containing 0.5% bovine serum albumin and 1 mM ethylenediaminetetraacetic acid, followed by separation by a cell sorting column at a flow rate of 1 mL / min. During washing, the sorting column was kept moist to avoid air bubbles. Positive cells were immediately resuspended in pre-cooled complete culture medium after elution. S5. Flow cytometry staining: The sorted immune cells are further analyzed and identified through live / dead staining, surface antibody staining, and resuspending of the cells to be tested. S6. Reagent kit assembly: Includes the special reagents and auxiliary materials from S1 to S5 above, and an instruction manual.

[0034] Comparative Example 3 The preparation method of the detection reagent for immune cells includes the following steps: S1. Tissue pretreatment: Fresh tissue is rinsed three times in pre-cooled sterile phosphate buffer to remove blood and necrotic parts, and then cut into 1-2 mm³ pieces with sterile ophthalmic scissors. Add pre-cooled phosphate buffer, let stand for 5 minutes, and aspirate the floating fat and blood vessel fragments on the surface. S2. Enzymatic digestion: A mixture of 1 mg / mL collagenase IV and 20 μg / mL deoxyribonuclease I was used. 0.1 mg / mL hyaluronidase was added to dense tissue. Hank's balanced salt solution containing Ca²⁺ / Mg²⁺ was used as the buffer. The mixture was digested in a shaker at 37°C for 60 minutes, with gentle pipetting every 10 minutes until the tissue block became flocculent. The reaction was then stopped, and an equal volume of DuPont modified Eagle medium containing 10% fetal bovine serum was added to terminate the enzyme activity. S3. Preparation of single-cell suspension: The cells were filtered sequentially through 100μm and 40μm cell sieves, rinsed with phosphate buffer three times, centrifuged at 300×g for 5 minutes, and the cell pellet was collected at 4℃. Cell viability was measured to be >90%. S4. Immunomagnetic bead sorting: Differentiate clusters of 4 microbeads per 10 μL of 10 μL. 7 The proportion of cells was determined by incubation in phosphate buffer containing 0.5% bovine serum albumin and 2 mM ethylenediaminetetraacetic acid, followed by separation by a cell sorting column at a flow rate of 1 mL / min. During washing, the sorting column was kept moist to avoid air bubbles. Positive cells were immediately resuspended in pre-cooled complete culture medium after elution. S5. Flow cytometry staining: The sorted immune cells are further analyzed and identified through live / dead staining, surface antibody staining, and resuspending of the cells to be tested. S6. Reagent kit assembly: Includes the special reagents and auxiliary materials from S1 to S5 above, and an instruction manual.

[0035] Comparative Example 4 The preparation method of the detection reagent for immune cells includes the following steps: S1. Tissue pretreatment: Fresh tissue is rinsed three times in pre-cooled sterile phosphate buffer to remove blood and necrotic parts, and then cut into 1-2 mm³ pieces with sterile ophthalmic scissors. Add pre-cooled phosphate buffer, let stand for 5 minutes, and aspirate the floating fat and blood vessel fragments on the surface. S2. Enzymatic digestion: A mixture of 1 mg / mL collagenase IV and 20 μg / mL deoxyribonuclease I was used. 0.1 mg / mL hyaluronidase was added to dense tissue. Hank's balanced salt solution containing Ca²⁺ / Mg²⁺ was used as the buffer. The mixture was digested in a shaker at 37°C for 60 minutes, with gentle pipetting every 10 minutes until the tissue block became flocculent. The reaction was then stopped, and an equal volume of DuPont modified Eagle medium containing 10% fetal bovine serum was added to terminate the enzyme activity. S3. Preparation of single-cell suspension: The cells were filtered sequentially through 100μm and 40μm cell sieves, rinsed with phosphate buffer three times, centrifuged at 300×g for 5 minutes, and the cell pellet was collected at 4℃. Cell viability was measured to be >90%. S4. Immunomagnetic bead sorting: Differentiate clusters of 4 microbeads per 10 μL of 10 μL. 7 The proportion of cells was determined by incubation in phosphate buffer containing 0.5% bovine serum albumin and 5 mM ethylenediaminetetraacetic acid, followed by separation by a cell sorting column at a flow rate of 1 mL / min. During washing, the sorting column was kept moist to avoid air bubbles. Positive cells were immediately resuspended in pre-cooled complete culture medium after elution. S5. Flow cytometry staining: The sorted immune cells are further analyzed and identified through live / dead staining, surface antibody staining, and resuspending of the cells to be tested. S6. Reagent kit assembly: Includes the special reagents and auxiliary materials from S1 to S5 above, and an instruction manual.

[0036] Comparative Examples 1, 2, 3, and 4 used the same CD4 microbeads (10 μL / 10 7 Cells), but the concentrations of EDTA in their buffer solutions were 0, 1, 2, and 5 mM (and all contained 0.5% BSA), as shown in Table 2: Table 2: Example EDTA concentration (mM) CD4+ cell recovery rate (%) Nonspecific binding (%) Comparative Example 1 0 65 ±4 12±2 Comparative Example 2 1 78±3 18±12 Comparative Example 3 2 92±2 3±0.55 Comparative Example 4 5 90±3 4±1 Conclusion: As shown in Table 2, the recovery rate was the highest (92%) and the non-specific binding rate was the lowest (3%) at 2 mM EDTA, which was better than other concentrations.

[0037] The terms "front," "back," "left," "right," "top," and "bottom" all refer to the figures in the accompanying drawings. Figure 1 Based on the perspective of the observer, the side of the device facing the observer is defined as the front, the left side of the observer is defined as the left, and so on.

[0038] In the description of this invention, it should be understood that the terms "center", "longitudinal", "lateral", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting the scope of protection of this invention.

[0039] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a reagent for detecting immune cells, characterized in that: Includes the following steps: S1. Tissue pretreatment: Fresh tissue is rinsed three times in pre-cooled sterile phosphate buffer and cut into 1-2 mm³ pieces with sterile ophthalmic scissors. S2. Enzymatic digestion: A mixture of 1-2 mg / mL collagenase IV and 20 μg / mL deoxyribonuclease I was used. 0.1 mg / mL hyaluronidase was added to dense tissue. Hank's balanced salt solution containing Ca²⁺ / Mg²⁺ was used as a buffer. The mixture was digested in a shaker at 37°C for 30-90 minutes. The reaction was stopped when the tissue block became flocculent by gentle pipetting. An equal volume of DuPont modified Eagle medium containing 10% fetal bovine serum was added to stop the enzyme activity. S3. Preparation of single-cell suspension: filter through 100μm and 40μm cell sieves in sequence, rinse the sieves 3 times with phosphate buffer, and centrifuge at 300×g for 5 minutes. S4. Immunomagnetic bead sorting: Differentiate clusters of 4 microbeads per 10 μL of 10 μL. 7 The proportion of cells was determined by incubation in phosphate buffer containing 0.5% bovine serum albumin and 2 mM ethylenediaminetetraacetic acid, followed by separation by a cell sorting column. Positive cells were eluted and immediately resuspended in pre-cooled complete culture medium. S5. Flow cytometry staining: The sorted immune cells are further analyzed and identified through live / dead staining, surface antibody staining, and resuspending of the cells to be tested. S6. Reagent kit assembly: Includes the special reagents and auxiliary materials from S1 to S5 above, and comes with an operation manual.

2. The method for preparing the immune cell detection reagent according to claim 1, characterized in that: The entire S1 step is performed on ice. If the tissue contains a large amount of blood in the S1 step, it should be treated with red blood cell lysis buffer for 5 minutes before washing. The preparation method of the red blood cell lysis buffer is as follows: Weigh 8.02g NH4Cl, 1.0g KHCO3, and 0.037g ethylenediaminetetraacetic acid-Na2, dissolve them in deionized water, bring the volume to 1L, adjust the pH to 7.2-7.4, filter to sterilize, and store at room temperature.

3. The method for preparing the immune cell detection reagent according to claim 1, characterized in that: The preparation method of the pre-cooled sterile phosphate buffer is as follows: Dissolve phosphate buffer powder in deionized water, adjust the pH to 7.2-7.4, filter to sterilize, and then pre-cool at 4°C for later use.

4. The method for preparing the immune cell detection reagent according to claim 1, characterized in that, The preparation method of the Duchenne modified Eagle medium containing 10% fetal bovine serum is as follows: Add 10% v / v fetal bovine serum to the basic Duchenne modified Eagle medium, mix well, filter to sterilize, and store in a 4°C refrigerator.

5. The method for preparing the immune cell detection reagent according to claim 1, characterized in that, The preparation method of the fetal bovine serum buffer containing 0.5% bovine serum albumin + 2mM ethylenediaminetetraacetic acid is as follows: Bovine serum albumin was dissolved in fetal bovine serum to achieve a bovine serum albumin concentration of 0.5% w / v. Then, ethylenediaminetetraacetic acid (EDTA) was added to achieve an EDTA concentration of 2 mM. After filtration and sterilization, the solution was stored at 4°C.

6. The method for preparing the immune cell detection reagent according to claim 1, characterized in that, The method for preparing the pre-cooled complete culture medium is as follows: Based on the growth requirements of the target immune cells, DuPont modified Eagle medium was selected, 10% to 20% v / v fetal bovine serum was added, and growth factors and antibiotics were added in proportion. After mixing evenly, the mixture was filtered to remove bacteria and placed in a 4°C refrigerator for pre-cooling before use. The mass ratio of collagenase IV to deoxyribonuclease I is 1:0.01–1:0.05; The differentiation cluster 4 microbeads are magnetic nanoparticles conjugated with anti-differentiation cluster 4 antibodies, with a particle size of 50-200 nm.

7. The method for preparing the immune cell detection reagent according to claim 1, characterized in that, The live / dead staining procedure: Take an appropriate amount of the sorted cell suspension, select the live or dead dye according to the experimental requirements, dilute it appropriately according to the operation manual before use, add an appropriate amount of live or dead dye, vortex mix, and incubate at 4°C for 30 minutes in the dark. Surface antibody staining procedure: Add fluorescently labeled antibodies against the target immune cell surface markers, vortex to mix, incubate at 4°C in the dark for 30 minutes, and wash the cells twice with flow cytometry buffer to remove unbound antibodies. Resuspension for inspection: Resuspend the cells in flow cytometry buffer, filter them before flow cytometry analysis, and prepare them for flow cytometry analysis.

8. The method for preparing the immune cell detection reagent according to claim 1, characterized in that, Before assembling the kit, each reagent and auxiliary material is subjected to quality testing, including testing for reagent purity, activity, and sterility, and testing for the integrity and sealing of auxiliary materials. The kit assembly includes: pre-cooled sterile phosphate buffer, enzymes, cell sieves, centrifuge tubes, magnetic beads, sorting columns, pre-cooled complete culture medium, flow cytometry-related antibodies, live / dead dyes, fixation / permeabilization kit, permeabilization solution, flow cytometry buffer, and an experimental operation manual. These components are assembled into the kit according to a reasonable layout and sequence. The operation manual includes the kit composition, usage methods, precautions, and result analysis.

9. A detection method for an immune cell detection reagent, applied to the preparation method of the immune cell detection reagent according to any one of claims 1-8, characterized in that, Includes the following steps: S1. Sample preparation: A suspension of differentiated cluster 4+ T cells was obtained after tissue pretreatment, enzymatic digestion, single-cell suspension preparation and immunomagnetic bead sorting. S2, Flow cytometry staining: S201. Live and dead staining: Take an appropriate amount of sorted cell suspension, select live and dead stain according to experimental requirements, dilute appropriately according to the operation manual before use, add an appropriate amount of live and dead stain, vortex mix, protect from light, and incubate at 4°C for 30 minutes. S202, Surface antibody staining: Add fluorescently labeled antibodies against the surface markers of the target immune cells, vortex to mix, incubate at 4°C in the dark for 30 minutes, wash the cells twice with flow cytometry buffer to remove unbound antibodies; S203, Resuspending cells for testing: Resuspend cells in flow cytometry buffer and filter them with a cell sieve before running the flow cytometer to remove cell clumps and prevent clogging. S204, Flow cytometry detection: Add the filtered cell suspension to the sample tube of the flow cytometer, set the detection parameters, start the flow cytometer to detect, collect and analyze the data, and judge the activity of immune cells and the expression of surface markers based on the fluorescence signal; S3. Results Analysis: Based on the flow cytometry results, and following the results analysis section of the kit's instruction manual, determine the relevant characteristics of immune cells, such as the ratio of live to dead cells and the expression level of surface markers. If abnormal results occur, handle them according to the solutions provided in the operation manual.

10. A detection reagent for immune cells, applied to the preparation method of the detection reagent for immune cells according to any one of claims 1-8, characterized in that, These include tissue processing reagents, enzymatic digestion reagents, single-cell suspension preparation reagents, immunomagnetic bead sorting reagents, and flow cytometry detection reagents.

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