Preparation method of B lymphocytes

Abstract

The invention relates to the field of bioengineering, in particular to a preparation method of B lymphocytes. The invention provides a preparation method of B lymphocytes. The preparation method comprises the following steps: S1, obtaining a single-cell suspension; s2, performing density gradient centrifugation on the single-cell suspension to obtain interface layer cells rich in mononuclear cells; s3, carrying out T cell lysis on the interface layer cells by adopting a specific antibody and a specific complement of an anti-T cell surface antigen, centrifuging, and collecting a precipitate; and S4, performing adherent purification on the cells obtained by collecting the precipitates in the step S3, and removing non-adherent cells to obtain the B lymphocytes. According to the extraction and purification method disclosed by the invention, researchers can efficiently enrich and obtain high-purity B lymphocyte populations from complex tissues under conventional laboratory conditions without purchasing professional sorting equipment, and the purity can meet strict requirements of various subsequent cell experiments and immune experiments.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and more particularly to a method for preparing B lymphocytes. Background Technology

[0002] B lymphocytes, as core effector cells of humoral immunity, play an irreplaceable role in antigen recognition, antibody secretion, and the formation of immune memory. They specifically bind to pathogen antigens through the B cell receptor (BCR), and upon activation, differentiate into plasma cells that secrete high-affinity antibodies, performing immune functions such as neutralizing pathogens, regulating opsonization, and activating the complement cascade. Simultaneously, B cells can differentiate into memory cells, providing long-term immune protection and laying the theoretical foundation for vaccine development. Therefore, obtaining high-purity primary B cells is crucial for research on immune mechanisms, analysis of the tumor microenvironment, and exploration of treatment strategies for autoimmune diseases.

[0003] Mice are the most widely used animal model for B cell research due to their highly conserved immune system (e.g., B cell differentiation pathways and surface marker expression profiles), clear genetic background, and low cost. C57BL / 6J mice have spleens rich in mature B cells (accounting for 10%-15% of total lymphocytes), including unique marginal zone B cells (MZ B cells). Their spleen structure is relatively loose, allowing for more complete cell release after mechanical grinding. Furthermore, the B cells within the mouse model exhibit uniform expression, stable CD19 expression, and weak complement resistance, making them an ideal sample source for studying tissue-specific immune responses. Primary B cells obtained through mouse models can provide direct experimental materials for understanding the pathogenesis of immune diseases and developing targeted drugs.

[0004] The spleen, as the largest peripheral lymphoid organ in the human body, is the core site for B cell differentiation and maturation. B cells are densely distributed in the primary follicles of the white pulp and the marginal zone of the red pulp (accounting for 55% of the total number of splenic lymphocytes), forming a spatial separation from T cells and providing an anatomical basis for targeted separation. However, the cellular heterogeneity of the spleen's microenvironment presents a dual challenge to B cell purification: spleen suspension contains a large number of T cells, macrophages, erythrocytes, and fibroblasts, and conventional density gradient centrifugation can only coarsely enrich mononuclear cells, resulting in insufficient B cell purity; an imbalance between activity and purity, such as strong lysis reagents (e.g., erythrocyte lysis buffer), can easily damage B cell surface markers, while gentle handling can lead to impurity residues; physical separation pressures (e.g., high-speed centrifugation, magnetic bead adsorption) may damage B cell activity, affecting subsequent antibody secretion capacity detection.

[0005] As a pivotal organ in the immune-metabolic system, the liver's unique immune microenvironment shapes specific subsets and functions of B cells. These B cells not only participate in hematopoiesis during embryonic development but also balance immune defense and tolerance in adulthood through unique subsets (activated B cells, Bregs). In chronic liver diseases, B cell dysregulation is a key factor driving hepatitis and fibrosis, while reversing this dysregulation signifies disease remission. Future precise regulation of intrahepatic B cell subsets will provide a new breakthrough in liver disease treatment.

[0006] Several cell isolation techniques have emerged to obtain high-purity primary B cells. However, existing isolation techniques have significant limitations: 1. Flow cytometry sorting relies on high-precision equipment (such as flow cytometers), requires specialized training, and has low cell recovery rates (loss rates of some subpopulations >30%); 2. Immunomagnetic bead sorting requires expensive antibody-conjugated magnetic beads (costing over a thousand yuan per sample), and strong magnetic fields can easily damage cell viability, affecting subsequent functional experiments; 3. Single density gradient centrifugation can only coarsely enrich mononuclear cells, resulting in insufficient B cell purity (often mixed with 20%-30% T cells / NK cells), making it difficult to meet the needs of high-precision research; 4. T cell clearance methods for isolating B lymphocytes are only suitable for B cell extraction from lymph node tissues, and have high antibody complement budgets, lacking universality for tissue and cell extraction. Summary of the Invention

[0007] In view of this, the present invention provides a method for preparing B lymphocytes. This invention provides an efficient, stable, and cost-effective experimental procedure specifically for the extraction and purification of primary B lymphocytes from the spleen and liver tissues of immunized mice. This innovative technique employs a three-step synergistic extraction and purification system: first, density gradient centrifugation; then, antibody-complement targeted selective lysis; and finally, differential adhesion treatment. The experimental procedures are clear, standardized, and easy to master. Furthermore, the entire process does not rely on expensive magnetic bead sorting systems or other specialized equipment, reducing research costs and providing a simpler and more cost-effective method for obtaining primary B lymphocytes. Using this extraction and purification method, researchers can efficiently enrich high-purity B lymphocyte populations from complex tissues under standard laboratory conditions without purchasing specialized sorting equipment. The purity meets the stringent requirements of subsequent cell and immunological experiments.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] This invention provides a method for preparing B lymphocytes, comprising the following steps:

[0010] S1: Obtain a single-cell suspension;

[0011] S2: Centrifuge the single-cell suspension using a density gradient to obtain an interface layer cell rich in mononuclear cells;

[0012] S3: The interface layer cells were lysed using specific antibodies against T cell surface antigens and specific complement, centrifuged, and the precipitate was collected;

[0013] S4: Collect the cells obtained from the precipitate in S3, purify them by adhering to the wall, remove the non-adherent cells, and obtain the B lymphocytes.

[0014] In some embodiments of the present invention, in the above preparation method, the density gradient centrifugation in S2 uses Ficoll lymphocyte separation medium.

[0015] In some embodiments of the present invention, the volume ratio of the Ficoll lymphocyte separation solution to the single-cell suspension in the above preparation method is 1:1.

[0016] In some embodiments of the present invention, the density of the Ficoll lymphocyte separation solution in the above preparation method is 1.083~1.085 g / mL.

[0017] In some embodiments of the present invention, the specific antibody in the above preparation method includes: CD90.2(Thy-1.2) antibody.

[0018] In some embodiments of the present invention, the specific complement in the above preparation method includes guinea pig complement.

[0019] In some embodiments of the present invention, the T cell lysis described in the above preparation method further includes: containing Ca 2+ Mg 2+ HBSS buffer.

[0020] In some embodiments of the present invention, the time for adhesion purification in the above preparation method is 45-65 min.

[0021] In some embodiments of the present invention, the single-cell suspension described in the above preparation method is derived from the liver or spleen.

[0022] The present invention also provides B lymphocytes obtained by the above preparation method.

[0023] In some embodiments of the present invention, the proportion of B220⁺ cells in the above-mentioned B lymphocytes is ≥90%.

[0024] The beneficial effects of this invention include:

[0025] This invention employs a multi-step synergistic purification technique, yielding mouse primary B lymphocytes with high purity and viability. Specifically, through an optimized multi-stage purification process, including but not limited to density gradient centrifugation, antibody-complement targeted lysis, and differential adhesion screening, highly efficient enrichment of primary cells is achieved. This method requires only a single mouse liver and spleen sampling to obtain a sufficient quantity of primary B lymphocytes, fully meeting the needs of subsequent cell culture and various identification experiments. The entire separation process strictly adheres to aseptic techniques and employs a closed operating system, minimizing the risk of exogenous contamination. Notably, the three-step synergistic purification strategy of this invention (density gradient centrifugation → antibody-complement targeted lysis → differential adhesion) forms a unique purification combination that effectively removes various impurity cells such as erythrocytes, granulocytes, and macrophages from tissues, significantly improving the yield and purity of the target primary B lymphocytes. Experimental data show that this method can stably obtain high-quality primary B cells with a purity of approximately 85% for spleen B cells, approximately 70% for liver B cells, and ≥90% viability within 4 hours. The entire process requires only conventional laboratory equipment such as a centrifuge and CO2 incubator, demonstrating excellent ease of operation and reproducibility. Finally, multi-parameter cell surface marker detection using flow cytometry and other methods confirmed that the primary B lymphocytes obtained by this method have superior identification performance and higher purity, providing reliable cell material for related research. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0027] Figure 1 This invention presents a flowchart of the extraction and purification process for B cells in an embodiment of the invention; wherein: the upper figure is a schematic diagram of the B cell extraction and purification process; the lower figure is a diagram showing the actual effect of the B cell extraction and purification process.

[0028] Figure 2 The diagram shows the purification results of B cells extracted from the spleen of C57BL / 6J mice in Example 1 of this invention; wherein: A shows the statistical results of the yield of B cells extracted and purified from the spleen of C57BL / 6J mice by flow cytometry using the unpurified lymphocytes obtained by Ficoll method and the method described above; B shows the statistical results of the apoptosis rate of B cells compared with the above method by flow cytometry; C shows the statistical results of the changes in B cell subset classification compared with the above method by flow cytometry; D shows the results of BCR activation compared with the above method by cell immunofluorescence detection.

[0029] Figure 3The diagram shows the purification results of B cells extracted from the spleen of C57BL / 6J mice in Comparative Example 1 of this invention; wherein: A shows the statistical results of B cell yield obtained from the spleen of C57BL / 6J mice after T cell elimination method used in the comparative example and purified by this method, as detected by flow cytometry; B shows the statistical results of B cell apoptosis rate compared with the above method, as detected by flow cytometry; C shows the statistical results of B cell subset classification changes compared with the above method, as detected by flow cytometry.

[0030] Figure 4 The diagram shows the purification results of B cells extracted from the liver of C57BL / 6J mice in Example 2 of this invention; wherein: A shows the statistical results of the yield of B cells extracted and purified from the liver of C57BL / 6J mice by Ficoll (unpurified method) and this method, as detected by flow cytometry; B shows the statistical results of the apoptosis rate of B cells compared with the above method, as detected by flow cytometry; C shows the statistical results of the changes in B cell subset classification compared with the above method, as detected by flow cytometry; D shows the results of BCR activation compared with the above method, as detected by cell immunofluorescence.

[0031] Figure 5 The diagram shows the purification results of B cells extracted from the spleen of BALB / c mice in Example 3 of this invention; wherein: A shows the statistical results of the yield of B cells extracted and purified from the spleen of BALB / c mice by Ficoll (unpurified method) and this method, as detected by flow cytometry; B shows the statistical results of the apoptosis rate of B cells compared with the above method, as detected by flow cytometry; C shows the statistical results of the changes in B cell subset classification compared with the above method, as detected by flow cytometry; D shows the results of BCR activation compared with the above method, as detected by cell immunofluorescence.

[0032] Figure 6 The diagram shows the purification results of B cells extracted from the liver of BALB / c mice in Example 4 of this invention; wherein: A shows the statistical results of the yield of B cells extracted and purified from the liver of BALB / c mice by Ficoll (unpurified method) and this method, as detected by flow cytometry; B shows the statistical results of the apoptosis rate of B cells compared with the above method, as detected by flow cytometry; C shows the statistical results of the changes in B cell subset classification compared with the above method, as detected by flow cytometry; D shows the results of BCR activation compared with the above method, as detected by cell immunofluorescence. Detailed Implementation

[0033] This invention discloses a method for preparing B lymphocytes.

[0034] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0035] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0036] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0037] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0038] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0039] The technical solution adopted in this invention is as follows: Through a multi-step synergistic strategy of reagent pretreatment, preparation of spleen single-cell suspension, density gradient centrifugation, selective clearance of T cells, adherent purification and flow cytometry identification, high-purity mouse primary liver and spleen B lymphocytes are isolated.

[0040] 1. Reagent pretreatment: 30 minutes before the experiment, place RPMI-1640 basal medium, Ficoll-Paque Premium 1.084 sterile solution (density range 1.084±0.001 g / mL), 1×HBSS balanced salt buffer, and 5% complete culture medium (RPMI-1640 medium containing 5% fetal bovine serum and 1% penicillin-streptomycin antibiotics) in a 37℃ constant temperature water bath to preheat the reagents to ensure that the reagents reach the optimal reaction temperature to maintain cell viability.

[0041] 2. Single-cell suspension preparation: 6-8 week old SPF-grade male C57BL / 6J mice were selected and euthanized by CO2 inhalation. The liver and spleen tissues were aseptically removed in a laminar flow hood. The spleen or liver was washed 2-3 times in pre-cooled sterile PBS buffer to remove peripheral blood contamination. The cells were then transferred to 60mm culture dishes containing 5% CM medium and gently ground with a sterile syringe plunger until the tissue was dispersed. The resulting cell suspension was filtered through a 100μm pore size cell filter membrane to thoroughly remove fibrous connective tissue and undispersed tissue fragments.

[0042] 3. Density gradient centrifugation: Slowly stack the filtered single-cell suspension and preheated Ficoll lymphocyte separation medium at a 1:1 volume ratio (5mL:5mL) into a 15mL centrifuge tube, maintaining a clear interface between the two phases. Use a horizontal rotor centrifuge, setting the centrifugation parameters to 1800 r / min, room temperature, for 20 minutes, with acceleration setting 9 and deceleration setting 1 (to avoid interface disturbance). After centrifugation, carefully aspirate the white membrane layer (rich in lymphocytes) located at the Ficoll-medium interface and perform viable cell counting using 0.4% trypan blue staining solution.

[0043] 4. Antibody-complement mediated T cell lysis: Calculate the amount of each reagent according to the formula for B cell positive screening: (1) 1×HBSS buffer volume (V1, mL) = total number of cells / (4×10) 7 (2) Complement volume (V2, mL) = V1 / 10, (3) Thy-1.2 antibody volume (V3, μL) = V1 (value) × 2. Then, the lymphocytes collected in step 3 were gently resuspended in preheated 1×HBSS buffer (volume V1), and guinea pig complement (volume V2) and anti-mouse CD90.2 (Thy-1.2) monoclonal antibody (volume V3) were added in proportion. After mixing, the mixture was incubated in a water bath at 37°C for 30 minutes. The mixture was gently mixed every 10 minutes to ensure sufficient reaction. Then, the mixture was centrifuged at 660×g for 5 minutes, and the supernatant was discarded to remove the lysed T cells.

[0044] 5. Adherent purification to remove monocytes: Resuspend the cell pellet in fresh 5% CM medium, transfer to a T75 cell culture flask, and incubate for 1 hour at 37℃, 5% CO2, and 95% humidity. Utilizing the non-adherent nature of B cells, gently pipette and collect the suspended cells in the culture flask, centrifuge at 660×g for 5 minutes, and the pellet is the preliminarily purified B lymphocytes.

[0045] In summary, the above steps show that Ficoll centrifugation can remove more than 90% of red blood cells and debris, making the subsequent T cell lysis system more stable, such as improving complement activity. Differential adhesion is achieved by the difference in adhesion rate between B cells and monocytes (B cell adhesion time > 60 minutes vs monocyte < 20 minutes) to achieve final purification.

[0046] 6. B cell purification and identification: The purified cells were used for phenotypic identification by flow cytometry. The specific steps were as follows: Cells were resuspended in PBS containing 2% FBS (1×10⁻⁶ cells / mL). 6 Add fluorescently labeled anti-mouse B220-BV605 monoclonal antibody (1:100 dilution) and isotype control antibody to 200 μL of cells. Incubate at 4°C in the dark for 30 minutes. After washing, load the sample onto a flow cytometer for detection. High-purity B lymphocytes are defined as those with a B220⁺ cell ratio ≥90%.

[0047] The reagents involved in this invention:

[0048] The preheating medium was RPMI-1640 medium, which was purchased commercially from Thermo Fisher Scientific Inc.

[0049] Preheated 1×HBSS buffer, commercially available from HyClone.

[0050] Preheated Ficoll density gradient solution, commercially available from Cytiva.

[0051] FBS for preheating, purchased commercially from Natocor (Natocor-Industria Biológica);

[0052] GUINEA PIG COMPLEMENT, commercially available from Rockland Immunochemicals;

[0053] Thy-1 (CD90.2), purchased from BioLegend.

[0054] Comparison of the present invention with the prior art:

[0055]

[0056] The technological innovations and advantages of this patented method are as follows: 1. Optimized density gradient centrifugation and reagent cost control: This patented method innovatively introduces density gradient centrifugation technology in the lymphocyte suspension preparation stage, significantly reducing cell density in the subsequent lysis step by pre-purifying lymphocytes. This "enrichment before lysis" strategy not only improves lysis efficiency but, more importantly, achieves the goal of scientifically adjusting the amount of antibody and complement used based on the actual cell quantity. Experimental data shows that this method can reduce reagent consumption by more than 50%, significantly reducing experimental costs while ensuring separation effectiveness. The application of density gradient centrifugation technology allows for optimal reagent ratios for samples of different sizes, avoiding resource waste or reagent shortages. 2. Differential adhesion technology for final purification: Addressing the limitation of existing technologies that do not address the removal of monocytes, this patented method innovatively employs differential adhesion technology as the final purification step. This method utilizes the differences in adhesion speed and characteristics of different cell types to selectively remove residual monocytes and dendritic cells by precisely controlling the culture time. Especially in the isolation of spleen B cells, differential adhesion technology can effectively remove fibroblasts and other monocytes with faster adhesion rates, significantly improving the purity of B cells. This physical separation method based on cell biology characteristics avoids cell damage that may be caused by chemical reagents, while maintaining the natural state of the cells. 3. Guarantee of cell viability and functional integrity: This patented method, through optimized separation procedures and operating conditions, can provide more stable and highly viable primary B cell samples. Functional verification experiments show that B cells extracted using this patented method fully retain the B cell receptor (BCR) signal transduction function, providing a high-quality cell model for subsequent immunological research. This maintenance of functional integrity is mainly attributed to the comprehensive consideration of gentle cell handling, reduced mechanical damage, and optimized culture conditions in the method. In particular, the differential adhesion step avoids excessive digestion or chemical treatment, maximizing the protection of the integrity of cell membrane surface molecules.

[0057] In summary, this patented method represents a systematic innovation and optimization in T-cell lysis technology, not only improving separation efficiency but also significantly broadening its applicability for extracting primary mouse B cells from various tissues. By organically combining density gradient centrifugation, antibody-complement targeted lysis, and differential adhesion technology, the entire process from sample processing to final purification is optimized. This provides high-purity, high-activity primary B cell resources for immunological research, thereby improving the reliability and reproducibility of related studies and promoting the in-depth development of basic immunological research and clinical applications.

[0058] In Examples 1 to 4 and Comparative Example 1 of this invention, all raw materials and reagents used can be purchased from the market.

[0059] The present invention will be further illustrated below with reference to the embodiments:

[0060] Example 1

[0061] Ten male mice aged 6-8 weeks (C57BL / 6J background) were obtained from Cyagen (Suzhou) Biotechnology Co., Ltd. They were housed in a standard specific pathogen-free (SPF) laboratory with five mice per cage, with free access to food and water. The room temperature was 20±2℃, humidity 55±5%, and light / dark cycle 12 hours. All experiments were conducted at the same time each day to avoid the influence of circadian rhythms. This study complied with the ethical standards of the International Association for the Care and Use of Animals (IACUC).

[0062] This embodiment details the entire process of extraction and purification of primary spleen B lymphocytes from C57BL / 6J mice. The specific experimental procedure is as follows: Figure 1 As shown.

[0063] 1. Preparatory work before the experiment includes: preheating and equilibrating the required experimental reagents in a 37℃ constant temperature water bath, including RPMI-1640 basal medium, Ficoll lymphocyte separation medium (density range 1.084±0.001 g / mL), and 1×HBSS buffer (containing Ca). 2+ Mg 2+ ), fetal bovine serum (FBS) and 5% complete culture medium.

[0064] 2. Ten male mice aged 6-8 weeks were euthanized, ensuring complete death. The mice were then disinfected by immersion in 75% ethanol and transferred to a clean bench for dissection. The skin and peritoneum were incised with sterile instruments, and the spleen was completely removed and placed in an EP tube on ice for preservation.

[0065] 3. Inside the biosafety cabinet, transfer the spleen from the EP tube to a 60mm sterile culture dish (culture dish a). Add 10mL of pre-chilled PBS buffer and gently rinse twice to thoroughly remove any hair and blood stains adhering to the spleen surface. Then transfer the cleaned spleen to a new sterile culture dish (culture dish b). Lay a 100μm pore size filter membrane flat on the tissue fragments, add 5mL of preheated 5% CM culture medium, and gently and thoroughly mechanically grind the cells using the flat end of a syringe plunger to obtain a cell suspension.

[0066] 4. Filter the cell suspension through a 100μm cell sieve into a 15mL centrifuge tube to remove undigested connective tissue and cell clumps. Centrifuge the filtrate at 660×g at room temperature for 5 minutes, carefully discard the supernatant, and gently resuspend the cell pellet in 5mL of 5% CM medium. Then, slowly stack the cell suspension onto an equal volume (5mL) of preheated Ficoll lymphocyte separation medium, taking care to maintain a clear interface between the two phases. Perform gradient centrifugation at 1800 r / min for 20 minutes (room temperature, speed 9 for ascending, speed 1 for descending). This step effectively separates cell populations of different densities.

[0067] 5. 15 minutes before the end of gradient centrifugation, remove the guinea pig complement reagent and CD90.2 (Thy-1.2) antibody from the refrigerator and place them on ice to thaw.

[0068] 6. After centrifugation, use a pipette to aspirate the white membrane layer at the Ficoll interface and transfer it to a new 15mL centrifuge tube. Add 5% CM culture medium to a final volume of 10mL, mix gently, and centrifuge at 660×g for 5 minutes to wash. Discard the supernatant, take a small amount of cell suspension and mix it with 0.4% trypan blue solution (1:1), and use a hemocytometer to count cells and detect cell viability.

[0069] 7. According to the experimental design, the cells were randomly divided into two groups (n=5 / group). The experimental data showed that the number of white membrane cells extracted from the spleens of 10 mice were as follows: Unpurified group (only Ficoll gradient centrifugation was performed to extract crude lymphocytes) ① 8.75×10 7 ②1.0×10 8 ③8.7×10 7 ④9.55×10 7 ⑤9.8×10 7 Purification group ⑥ 8.2×10 7 ⑦9.2×10 7 ⑧1.02×10 8 ⑨8.6×10 7 ⑩8.56×10 7 The unpurified group was directly taken at a rate of 1×10⁻⁶. 6 Cells were subjected to flow cytometry staining; the purified group underwent B-cell positive screening using the following formula, with the required amounts of each reagent calculated accordingly:

[0070] (1) 1×HBSS buffer volume (V1 mL) = total cell number / (4×10) 7 );

[0071] (2) Complement volume (V2 mL) = V1 / 10;

[0072] (3) Thy-1.2 antibody volume (V3 μL) = V1 (numerical value) × 2

[0073] The required reagent volumes for each sample in the purification group were calculated as follows: ⑥ V1 = 2.05 mL, V2 = 0.205 mL, V3 = 4.1 μL; ⑦ V1 = 2.30 mL, V2 = 0.230 mL, V3 = 4.6 μL; ⑧ V1 = 2.55 mL, V2 = 0.255 mL, V3 = 5.1 μL; ⑨ V1 = 2.15 mL, V2 = 0.215 mL, V3 = 4.3 μL; ⑩ V1 = 2.14 mL, V2 = 0.214 mL, V3 = 4.28 μL.

[0074] 8. Incubate the prepared cell-antibody-complement mixture in a 37°C water bath for 30 minutes, gently inverting and mixing every 10 minutes. After incubation, add 1×HBSS buffer to a final volume of 10 mL and centrifuge at 660×g for 5 minutes. At this point, T cells are lysed by complement due to the binding of the Thy-1.2 antigen and antibody on their surface, while B cells and monocytes precipitate at the bottom of the tube.

[0075] 9. After discarding the supernatant, resuspend the cell pellet in 5 mL of 5% CM and transfer the suspension to a T75 culture flask. Wash the centrifuge tube twice with 5 mL of culture medium, and combine all the liquids into the same culture flask to a final volume of 15 mL. Incubate the culture flask in a 37°C, 5% CO2 incubator for 1 hour to allow the monocytes to adhere. Then collect the non-adherent cell suspension, centrifuge at 660×g for 5 minutes to obtain an enriched B cell population.

[0076] 10. Flow cytometry analysis used the following antibody combination: total B cell marker was Anti-mouse-B220-BV605; apoptosis detection used 7-AAD-Percp + Annexin V-FITC; B cell subset markers were CD23-PE + CD21-PE-Cy7. 1×10⁻⁶ samples were taken from each sample. 6 Cells were incubated with antibody suspension (50 μL) for 30 minutes in the dark, followed by washing twice with PBS and resuspending in 200 μL PBS. Data were acquired using a Longcyte C3140 flow cytometer and Modeflower software, and the results were analyzed using FlowJo v10.9.0 software. Specific results are shown below. Figure 2 As shown.

[0077] 11. Using a cell counting chamber, lymphocyte suspensions obtained after density gradient centrifugation of the unpurified and purified groups were added to count the cells. The mean number of unpurified white membrane cells was (9.36±0.602)×10⁻⁶. 7 The mean number of white membrane cells in the purified group was (8.95±0.785)×10⁻⁶. 7 The mean values ​​of the two sets of data showed no significant difference, allowing for comparison during subsequent purification operations.

[0078] 12. B-cell yield determination: Total B-cell markers were detected by flow cytometry as described above. The results showed that the B-cell yield in the unpurified group was 54.46±3.347%; the B-cell yield in the purified group was significantly increased to 90.92±1.632%. The difference between the two groups was statistically significant. Specific results are as follows: Figure 2As shown in Figure A, this purification process effectively removes non-target cells such as T cells and macrophages, thereby efficiently enriching B cells.

[0079] 13. Apoptosis Rate and Viability Identification: Using the flow cytometry method described above, the apoptosis rate of the unpurified group was 0.056 ± 0.011%; the apoptosis rate of the purified group decreased significantly to 0.005 ± 0.002%. The difference between the two groups was statistically significant. See below for details. Figure 2 The purification process did not induce additional apoptosis; instead, it reduced the apoptosis level, possibly due to the removal of apoptotic cells, such as senescent or damaged cells. High-purity B cells exhibit more stable activity, which is beneficial for subsequent functional experiments, such as in vitro culture or stimulation experiments.

[0080] 14. Identification of B-cell subset proportions: B-cell subset markers were detected by flow cytometry as described above. The results showed that: the proportion of follicular B cells (FO B cells) in the unpurified group was 73.86±2.011%, while the proportion in the purified group was 81.44±1.531%; the proportion of marginal zone B cells (MZ B cells) in the unpurified group was 15.94±2.024%, while the proportion in the purified group was 10.95±0.917%; the proportion of B1 B cells in the unpurified group was 4.508±0.849%, while the proportion in the purified group was 3.562±0.522%. See below for details. Figure 2 The results show that the proportion of FO B cells increased and the proportions of MZ B cells and B1 B cells decreased after purification. However, the changes in various B cell subsets in both the unpurified and purified groups were within the normal range and did not affect the overall B cell subset classification. Therefore, these results provide a high-purity, high-activity cellular basis for subsequent B cell function studies, such as antibody secretion and signaling pathway regulation.

[0081] 15. Immunofluorescence Detection: Images of unpurified B cells and primary B cells prepared using a Nikon A1R laser confocal microscope were acquired. B cells were activated with free antigens, and changes resulting from antigen receptor (BCR) activation were detected. The images were processed and analyzed using NIS element software to assess whether the activation function of the primary B cells extracted using this method had been altered. Image results are shown below. Figure 2 As shown in Figure D, the changes in the surface of B cells extracted and purified by this method over time are within the normal range, confirming that the integrity of the extracted cell membrane proteins and their signal transduction function are normal, indicating that the B cells extracted by this method can be used for subsequent immunological research.

[0082] Table 1

[0083]

[0084] Comparative Example 1

[0085] The T-cell clearance method is a currently disclosed method for isolating B lymphocytes by clearing T cells. The principle of this method is as follows: T cells are bound with anti-T-cell monoclonal antibodies, and complement-mediated cytotoxicity causes T-cell lysis. Dead cells are then removed using Ficoll gradient density centrifugation to collect the B cells. This comparative example details the entire process of isolating primary spleen B lymphocytes from C57BL / 6J mice using the existing T-cell clearance method and compares it with the results of this patent. The specific steps include:

[0086] 1. Preparation of Single-Cell Suspension: The specific steps for preparing the spleen lymphocyte suspension are basically the same as steps 1-6 in Example 1. Lymphocytes are separated by density gradient centrifugation. After centrifugation, the white membrane layer at the Ficoll interface is aspirated and transferred to a new 15mL centrifuge tube. An appropriate volume of culture medium is added, and the mixture is gently mixed and then centrifuged at 660×g for 5 minutes for washing. The centrifuged lymphocyte pellet is resuspended in 0.5ml of 5% CM to obtain the lymphocyte suspension.

[0087] 2. Lysis of T cells: In 10 tubes of prepared lymphocyte suspension, add 0.5 mL of anti-mouse T cell monoclonal antibody to each tube at a ratio of 10:1 (volume ratio) to mix the cell suspension. Incubate at 37°C for 30 minutes. After incubation, add 125 μL of guinea pig complement to each tube at a ratio of 4:1 (volume ratio) to obtain a mixed antibody and complement cell suspension. Incubate at 37°C for another 30 minutes to induce 100% lysis of T cells. After incubation, add a quantitative amount of sterile PBS to each tube and wash once by centrifugation. Then resuspend the cells in PBS.

[0088] 3. Ficoll method for purifying B cells: Centrifuge for 20 minutes using Ficoll gradient density centrifugation, discard the dead T cells in the first white ring layer, carefully collect the live cells (B cells) in the second white ring layer, wash once with sterile PBS, and then resuspend in RPMI 1640 culture medium containing 10% FBS to obtain B lymphocytes obtained by T cell clearance method.

[0089] 4. The B lymphocytes obtained by the T-cell clearance method used in the comparative example were identified by flow cytometry, and the results were compared and analyzed with the B lymphocytes extracted and purified by the method of this patent in Example 1. The analysis is as follows:

[0090] 5. The antibody combination and specific steps used in flow cytometry analysis were consistent with those in Example 1. A Longcyte C3140 flow cytometer and Modeflower software were used to collect data, and FlowJo v10.9.0 software was used to analyze the results. Specific results are shown below. Figure 3 As shown.

[0091] 6. B-cell yield identification: Total B-cell markers were detected by flow cytometry as described above. The results showed that the B-cell yield of this patented method was significantly increased to 90.92±1.632%; the B-cell yield of the comparative method was 56.8±6.106%. The difference between the two groups was statistically significant. Specific results are as follows: Figure 3 As shown in A, this patented method achieves superior B cell extraction results using less reagent during the extraction process, indicating that it can more effectively remove non-target cells such as T cells and macrophages, thereby efficiently enriching B cells.

[0092] 7. Apoptosis Rate and Viability Identification: Using flow cytometry to detect apoptotic cells, the apoptosis rate of this patented method was significantly reduced to 0.005±0.002%; the apoptosis rate of the comparative method was 0.044±0.012%. The difference between the two groups was statistically significant. See below for details. Figure 3 The high-purity B cells extracted by this patented method exhibit more stable cell activity compared to existing T-cell clearance methods, which is beneficial for subsequent immunological experiments.

[0093] 8. Identification of B-cell subset proportions: B-cell subset markers were detected by flow cytometry. The results showed that: in this patented method, the proportion of follicular B cells (FO B cells) was 81.44±1.531%, while in the comparative method, the proportion of this cell type was 49.85±10.57%; in this patented method, the proportion of marginal zone B cells (MZ B cells) was 10.95±0.917%, while in the comparative method, the proportion was 16.77±2.096%; in this patented method, the proportion of B1 B cells was 3.562±0.522%, while in the comparative method, the proportion was 14.89±3.879%. For detailed results, see [link to results]. Figure 3Studies have shown that FO B cells account for approximately 70-80% of spleen B cells, MZB cells for approximately 10-20%, while the proportion of B1 cells is extremely low, typically less than 5%. Comparative analysis revealed that the proportions of MZ B cells and B1 B cells increased in the B cells extracted using the method mentioned in the comparison, while the proportion of FO B cells decreased accordingly. Although the increase in the proportion of MZ B cells was within the normal range, the increase in the proportion of B1 B cells and the decrease in the proportion of FO B cells both exceeded the normal fluctuation range. Therefore, the proportions of each subpopulation in primary B cells extracted using this comparative method were significantly altered, indicating that the cells extracted using this method are not stable enough to meet the demand for high-quality and stable primary B cell samples for subsequent B cell function studies.

[0094] Table 2

[0095]

[0096] Technical limitations of the comparative method: 1. Limited tissue applicability: The T-cell lysis method used in the comparative method directly lyses T cells after preparing a single-cell suspension. This method has significant limitations in terms of tissue applicability. Studies have shown that this method is only suitable for tissue samples with relatively simple components, such as lymph nodes. The separation effect is significantly reduced for complex tissues with multiple cell components, such as the spleen and liver. This is mainly because intercellular interactions and matrix components in complex tissues interfere with the uniform distribution of lysis reagents, leading to uneven lysis efficiency, which in turn affects the yield and purity of B cells. 2. Low reagent cost-effectiveness: In the T-cell lysis step, the comparative method adopts a strategy of processing a uniform cell suspension volume without dynamically adjusting the reagent dosage according to the actual cell number, resulting in low reagent utilization efficiency. Specifically, this method uses a fixed amount of antibody and complement reagents regardless of the cell number in the sample, causing two extreme situations: for low cell number samples, there is a serious waste of reagents; for high cell number samples, insufficient reagents may lead to incomplete lysis. This untargeted reagent utilization strategy significantly increases experimental costs and does not meet the cost-effectiveness requirements of modern biomedical research. 3. Incomplete purification: The comparative method did not effectively separate B cells from other monocytes after T cell lysis, resulting in a significant proportion of impurities such as monocytes and dendritic cells remaining in the final product. The presence of these contaminating cells not only reduces the purity of B cells but may also affect the biological behavior of B cells through cell-cell interactions, potentially leading to biased results, especially in subsequent functional experiments.

[0097] Example 2

[0098] Ten male mice aged 6-8 weeks (C57BL / 6J background) were obtained from Cyagen (Suzhou) Biotechnology Co., Ltd. They were housed in a standard specific pathogen-free (SPF) laboratory with five mice per cage, with free access to food and water. The room temperature was 20±2℃, humidity 55±5%, and light / dark cycle 12 hours. All experiments were conducted at the same time each day to avoid the influence of circadian rhythms. This study complied with the ethical standards of the International Association for the Care and Use of Animals (IACUC).

[0099] This embodiment details the entire process of extraction and purification of primary liver B lymphocytes from C57BL / 6J mice. The specific experimental procedure is as follows: Figure 1 As shown.

[0100] 1. Preparatory work before the experiment includes: preheating and equilibrating the required experimental reagents in a 37℃ constant temperature water bath, including RPMI-1640 basal medium, Ficoll lymphocyte separation medium (density range 1.084±0.001 g / mL), and 1×HBSS buffer (containing Ca). 2+ Mg 2+ ), fetal bovine serum (FBS) and 5% complete culture medium.

[0101] 2. Ten male mice aged 6-8 weeks were euthanized, ensuring complete death. The mice were then disinfected by immersion in 75% ethanol and transferred to a laminar flow hood for dissection. The skin and peritoneum were incised using sterile instruments, and the liver was completely removed and placed in an EP tube for preservation on ice.

[0102] 3. Inside the biosafety cabinet, transfer the liver from the EP tube to a 60mm sterile culture dish (culture dish a). Add 10mL of pre-chilled PBS buffer and gently rinse twice to thoroughly remove any hair and bloodstains adhering to the liver surface. Then transfer the cleaned liver to a new sterile culture dish (culture dish b). Spread a 100μm pore size filter membrane evenly on the tissue fragments, add 5mL of preheated 5% CM culture medium, and gently and thoroughly mechanically grind using the flat end of a syringe plunger to obtain a cell suspension.

[0103] 4. Filter the cell suspension through a 100μm cell sieve into a 15mL centrifuge tube to remove undigested connective tissue and cell clumps. Centrifuge the filtrate at 660×g at room temperature for 5 minutes, carefully discard the supernatant, and gently resuspend the cell pellet in 5mL of 5% CM medium. Then, slowly stack the cell suspension onto an equal volume of preheated Ficoll lymphocyte separation medium, taking care to maintain a clear interface between the two phases. Perform gradient centrifugation at 1800 r / min for 20 minutes (room temperature, speed 9 for ascending, speed 1 for descending). This step effectively separates cell populations of different densities.

[0104] 5. 15 minutes before the end of gradient centrifugation, remove the guinea pig complement reagent and CD90.2 (Thy-1.2) antibody from the refrigerator and place them on ice to thaw.

[0105] 6. After centrifugation, use a pipette to aspirate the white membrane layer at the Ficoll interface and transfer it to a new 15mL centrifuge tube. Add 5% CM culture medium to a final volume of 10mL, mix gently, and centrifuge at 660×g for 5 minutes to wash. Discard the supernatant, take a small amount of cell suspension and mix it with 0.4% trypan blue solution (1:1), and use a hemocytometer to count cells and detect cell viability.

[0106] 7. According to the experimental design, the cells were randomly divided into two groups (n=5 / group). The experimental data showed that the number of white blood cell layers extracted from the livers of 10 mice was as follows: Unpurified group ① 2.5×10 6 ②3.1×10 6 ③4.2×10 6 ④3.4×10 6 ⑤3.8×10 6 Purification group ⑥ 2.4×10 6 ⑦3.9×10 6 ⑧2.7×10 6 ⑨3.3×10 6 ⑩4.0×10 6 The unpurified group was directly taken at a rate of 1×10⁻⁶. 6 Cells were subjected to flow cytometry staining; the purified group underwent B-cell positive screening using the following formula, with the required amounts of each reagent calculated accordingly:

[0107] (1) 1×HBSS buffer volume (V1 mL) = total cell number / (4×10) 7 );

[0108] (2) Complement volume (V2 mL) = V1 / 10;

[0109] (3) Thy-1.2 antibody volume (V3 μL) = V1 (numerical value) × 2;

[0110] 8. The required reagent volumes for each sample in the purification group were calculated as follows: ⑥ V1 = 0.060 mL, V2 = 0.006 mL, V3 = 0.12 μL ⑦ V1 = 0.098 mL, V2 = 0.010 mL, V3 = 0.195 μL ⑧ V1 = 0.068 mL, V2 = 0.007 mL, V3 = 0.136 μL ⑨ V1 = 0.083 mL, V2 = 0.008 mL, V3 = 0.165 μL ⑩ V1 = 0.100 mL, V2 = 0.010 mL, V3 = 0.200 μL;

[0111] 9. Incubate the prepared cell-antibody-complement mixture in a 37°C water bath for 30 minutes, gently inverting and mixing every 10 minutes. After incubation, add 1×HBSS buffer to a final volume of 10 mL and centrifuge at 660×g for 5 minutes. At this point, T cells will be lysed by complement due to the binding of the Thy-1.2 antigen and antibody on their surface, while B cells and monocytes will precipitate at the bottom of the tube.

[0112] 10. After discarding the supernatant, resuspend the cell pellet in 5 mL of 5% CM and transfer the suspension to a T75 culture flask. Wash the centrifuge tube twice with 5 mL of culture medium, and combine all the liquids into the same culture flask to make a final volume of 15 mL. Incubate the culture flask in a 37°C, 5% CO2 incubator for 1 hour to allow the monocytes to adhere. Then collect the non-adherent cell suspension, centrifuge at 660×g for 5 minutes to obtain an enriched B cell population.

[0113] 11. Flow cytometry analysis used the following antibody combination: total B cell marker was Anti-mouse-B220-BV605; apoptosis detection used 7-AAD-Percp + Annexin V-FITC; B cell subset markers were CD23-PE + CD21-PE-Cy7. 1×10⁻⁶ samples were taken from each sample. 6 Cells were incubated with antibody suspension (50 μL) for 30 minutes in the dark, followed by washing twice with PBS and resuspending in 200 μL PBS. Data were acquired using a Longcyte C3140 flow cytometer and Modeflower software, and the results were analyzed using FlowJo v10.9.0 software. Specific results are shown below. Figure 4 As shown.

[0114] 12. Cell counting was performed using a cell counting chamber by adding lymphocyte suspensions obtained from density gradient centrifugation of the unpurified and purified groups. The mean number of unpurified white membrane cells was (3.4±0.652)×10⁻⁶. 6 The mean number of white membrane cells in the purified group was (3.26±0.709)×10⁻⁶. 6 The mean values ​​of the two sets of data showed no significant difference, allowing for comparison during subsequent purification operations.

[0115] 13. B-cell yield determination: Total B-cell markers were detected by flow cytometry as described above. The results showed that the B-cell yield in the unpurified group was 25.56±2.458%, while the B-cell yield in the purified group was significantly increased to 71.3±3.973%. The difference between the two groups was statistically significant. Specific results are as follows: Figure 4 As shown in A.

[0116] 14. Apoptosis Rate and Viability Identification: The results obtained using the flow cytometry method for detecting apoptotic cells showed that the apoptosis rate in the unpurified group was 0.811±0.646%; the apoptosis rate in the purified group significantly decreased to 0.020±0.022%. The difference between the two groups was statistically significant. Specific results are as follows: Figure 4 As shown in B.

[0117] 15. Identification of B-cell subset proportions: B-cell subset markers were detected by flow cytometry as described above. The results showed that the proportion of follicular B cells in the unpurified group was 84.64±2.768%, while the proportion in the purified group was 87.58±1.219%; the proportion of B1 B cells in the unpurified group was 4.832±1.516%, while the proportion in the purified group was 3.792±0.755%. See below for details. Figure 4 C. This indicates that after purification, the proportion of FOB cells increased and the proportion of B1 B cells decreased. Furthermore, the various B cell subsets in both the unpurified and purified groups fluctuated within the normal range and did not affect the overall B cell subset classification. This provides a high-purity and high-activity cellular basis for subsequent B cell function studies.

[0118] 16. Immunofluorescence Detection: Images of unpurified B cells and primary B cells prepared using a Nikon A1R laser confocal microscope were acquired. B cells were activated with free antigens, and changes resulting from antigen receptor (BCR) activation were detected. The images were processed and analyzed using NIS element software to assess whether the activation function of the primary B cells extracted using this method was altered. Image results are shown below. Figure 4 As shown in Figure D, the changes in the surface of B cells extracted and purified by this method over time are within the normal range, confirming that the integrity of the extracted cell membrane proteins and their signal transduction function are normal, indicating that the B cells extracted by this method can be used for subsequent immunological research.

[0119] Table 3

[0120]

[0121] Example 3

[0122] Ten male 6-8 week old BALB / c mice, sourced from Cyagen (Suzhou) Biotechnology Co., Ltd., were housed in a standard specific pathogen-free (SPF) laboratory with five mice per cage. Mice had free access to food and water, and the room temperature was 20±2℃, humidity 55±5%, with a 12-hour light / dark cycle. All experiments were conducted at the same time each day to avoid the influence of circadian rhythms. This study complied with the ethical standards of the International Association for the Care and Use of Animals (IACUC).

[0123] This embodiment details the entire process of extraction and purification of primary spleen B lymphocytes from BALB / c mice. The specific experimental procedure is as follows: Figure 1 As shown.

[0124] 1. Preparatory work before the experiment includes: preheating and equilibrating the required experimental reagents in a 37℃ constant temperature water bath, including RPMI-1640 basal medium, Ficoll lymphocyte separation medium (density range 1.084±0.001 g / mL), and 1×HBSS buffer (containing Ca). 2+ Mg 2+ ), fetal bovine serum (FBS) and 5% complete culture medium.

[0125] 2. Ten male mice aged 6-8 weeks were euthanized, ensuring complete death. The mice were then disinfected by immersion in 75% ethanol and transferred to a clean bench for dissection. The skin and peritoneum were incised with sterile instruments, and the spleen was completely removed and placed in an EP tube on ice for preservation.

[0126] 3. Inside the biosafety cabinet, transfer the spleen from the EP tube to a 60mm sterile culture dish (culture dish a). Add 10mL of pre-chilled PBS buffer and gently rinse twice to thoroughly remove any hair and blood stains adhering to the spleen surface. Then transfer the cleaned spleen to a new sterile culture dish (culture dish b). Lay a 100μm pore size filter membrane flat on the tissue fragments, add 5mL of preheated 5% CM culture medium, and gently and thoroughly mechanically grind the cells using the flat end of a syringe plunger to obtain a cell suspension.

[0127] 4. Filter the cell suspension through a 100μm cell sieve into a 15mL centrifuge tube to remove undigested connective tissue and cell clumps. Centrifuge the filtrate at 660×g at room temperature for 5 minutes, carefully discard the supernatant, and gently resuspend the cell pellet in 5mL of 5% CM medium. Then, slowly stack the cell suspension onto an equal volume (5mL) of preheated Ficoll lymphocyte separation medium, taking care to maintain a clear interface between the two phases. Perform gradient centrifugation at 1800 r / min for 20 minutes (room temperature, speed 9 for ascending, speed 1 for descending). This step effectively separates cell populations of different densities.

[0128] 5. 15 minutes before the end of gradient centrifugation, remove the guinea pig complement reagent and CD90.2 (Thy-1.2) antibody from the refrigerator and place them on ice to thaw.

[0129] 6. After centrifugation, use a pipette to aspirate the white membrane layer at the Ficoll interface and transfer it to a new 15mL centrifuge tube. Add 5% CM culture medium to a final volume of 10mL, mix gently, and centrifuge at 660×g for 5 minutes to wash. Discard the supernatant, take a small amount of cell suspension and mix it with 0.4% trypan blue solution (1:1), and use a hemocytometer to count cells and detect cell viability.

[0130] 7. According to the experimental design, the cells were randomly divided into two groups (n=5 / group). The experimental data showed that the number of white membrane cells extracted from the spleens of 10 mice were as follows: Unpurified group ① 6.77×10 7 ②7.69×10 7 ③8.27×10 7 ④6.93×10 7 ⑤7.21×10 7 Purification group ⑥ 7.66×10 7 ⑦6.84×10 7 ⑧6.56×10 7 ⑨7.83×10 7 ⑩7.74×10 7 The unpurified group was directly taken at a rate of 1×10⁻⁶. 6 Cells were subjected to flow cytometry staining; the purified group underwent B-cell positive screening using the following formula, with the required amounts of each reagent calculated accordingly:

[0131] (1) 1×HBSS buffer volume (V1 mL) = total cell number / (4×10) 7 );

[0132] (2) Complement volume (V2 mL) = V1 / 10;

[0133] (3) Thy-1.2 antibody volume (V3 μL) = V1 (numerical value) × 2;

[0134] 8. The required reagent volumes for each sample in the purification group were calculated as follows: ⑥ V1 = 1.915 mL, V2 = 0.192 mL, V3 = 3.83 μL; ⑦ V1 = 1.71 mL, V2 = 0.171 mL, V3 = 3.42 μL; ⑧ V1 = 1.64 mL, V2 = 0.164 mL, V3 = 3.28 μL; ⑨ V1 = 1.958 mL, V2 = 0.196 mL, V3 = 3.915 μL; ⑩ V1 = 1.935 mL, V2 = 0.194 mL, V3 = 3.87 μL.

[0135] 9. Incubate the prepared cell-antibody-complement mixture in a 37°C water bath for 30 minutes, gently inverting and mixing every 10 minutes. After incubation, add 1×HBSS buffer to a final volume of 10 mL and centrifuge at 660×g for 5 minutes. At this point, T cells will be lysed by complement due to the binding of the Thy-1.2 antigen and antibody on their surface, while B cells and monocytes will precipitate at the bottom of the tube.

[0136] 10. After discarding the supernatant, resuspend the cell pellet in 5 mL of 5% CM and transfer the suspension to a T75 culture flask. Wash the centrifuge tube twice with 5 mL of culture medium, and combine all the liquids into the same culture flask to a final volume of 15 mL. Incubate the culture flask in a 37°C, 5% CO2 incubator for 1 hour to allow the monocytes to adhere. Then collect the non-adherent cell suspension, centrifuge at 660×g for 5 minutes to obtain an enriched B cell population.

[0137] 11. Flow cytometry analysis used the following antibody combination: total B cell marker was Anti-mouse-B220-BV605; apoptosis detection used 7-AAD-Percp + Annexin V-FITC; B cell subset marker was CD23-PE + CD21-PE-Cy7. 1×10⁻⁶ samples were taken from each sample. 6 Cells were incubated with antibody suspension (50 μL) for 30 minutes in the dark, followed by washing twice with PBS and resuspending in 200 μL PBS. Data were acquired using a Longcyte C3140 flow cytometer and Modeflower software, and the results were analyzed using FlowJo v10.9.0 software. Specific results are shown below. Figure 5 As shown.

[0138] 12. Cell counting was performed using a cell counting chamber by adding lymphocyte suspensions obtained from density gradient centrifugation of the unpurified and purified groups. The mean number of unpurified white membrane cells was (7.374±0.611)×10⁻⁶. 7 The mean number of white membrane cells in the purified group was (7.326±0.583)×10⁻⁶. 7 The mean values ​​of the two sets of data showed no significant difference, allowing for comparison during subsequent purification operations.

[0139] 13. B-cell yield identification: Total B-cell markers were detected by flow cytometry as described above. The results showed that the B-cell yield in the unpurified group was 43.68±4.539%; the B-cell yield in the purified group was significantly increased to 54.6±4.056%. The difference between the two groups was statistically significant. Specific results are as follows: Figure 5As shown in Figure A, this purification process effectively removes non-target cells such as T cells and macrophages, thereby efficiently enriching B cells.

[0140] 14. Apoptosis Rate and Viability Identification: Using the flow cytometry method described above, the apoptosis rate of the unpurified group was 0.264±0.099%; the apoptosis rate of the purified group was significantly reduced to 0.021±0.004%. The difference between the two groups was statistically significant. See below for details. Figure 5 The purification process did not induce additional apoptosis; instead, it reduced the apoptosis level, possibly due to the removal of apoptotic cells, such as senescent or damaged cells. High-purity B cells exhibit more stable activity, which is beneficial for subsequent functional experiments, such as in vitro culture or stimulation experiments.

[0141] 15. Identification of B cell subset proportions: B cell subset markers were detected by flow cytometry as described above. The results showed that: the proportion of follicular B cells (FO B cells) in the unpurified group was 72.22±0.589%, while the proportion in the purified group was 76.82±2.547%; the proportion of marginal zone B cells (MZ B cells) in the unpurified group was 7.566±0.516%, while the proportion in the purified group was 6.814±0.620%; the proportion of B1 B cells in the unpurified group was 12.88±1.299%, while the proportion in the purified group was 10.00±1.883%. See below for detailed results. Figure 5 The results show that the proportion of FO B cells increased and the proportions of MZ B cells and B1 B cells decreased after purification. However, the changes in various B cell subsets in both the unpurified and purified groups were within the normal range and did not affect the overall B cell subset classification. Therefore, these results provide a high-purity, high-activity cellular basis for subsequent B cell function studies, such as antibody secretion and signaling pathway regulation.

[0142] 16. Immunofluorescence Detection: Images of unpurified B cells and primary B cells prepared using a Nikon A1R laser confocal microscope were acquired. B cells were activated with free antigens, and changes resulting from antigen receptor (BCR) activation were detected. The images were processed and analyzed using NIS element software to assess whether the activation function of the primary B cells extracted using this method was altered. Image results are shown below. Figure 5 As shown in Figure D, the changes in the surface of B cells extracted and purified by this method over time are within the normal range, confirming that the integrity of the extracted cell membrane proteins and their signal transduction function are normal, indicating that the B cells extracted by this method can be used for subsequent immunological research.

[0143] Table 4

[0144]

[0145] Example 4

[0146] Ten male mice aged 6-8 weeks, with a BALB / c background, were obtained from Cyagen (Suzhou) Biotechnology Co., Ltd. They were housed in a standard specific pathogen-free (SPF) laboratory with five mice per cage, with free access to food and water. The room temperature was 20±2℃, humidity 55±5%, and the light / dark cycle was 12 hours. All experiments were conducted at the same time each day to avoid the influence of circadian rhythms. This study complied with the ethical standards of the International Association for the Care and Use of Animals (IACUC).

[0147] This embodiment details the entire process of extraction and purification of primary liver B lymphocytes from BALB / c mice. The specific experimental procedure is as follows: Figure 1 As shown.

[0148] 1. Preparatory work before the experiment includes: preheating and equilibrating the required experimental reagents in a 37℃ constant temperature water bath, including RPMI-1640 basal medium, Ficoll lymphocyte separation medium (density range 1.084±0.001 g / mL), and 1×HBSS buffer (containing Ca). 2+ Mg 2+ ), fetal bovine serum (FBS) and 5% complete culture medium.

[0149] 2. Ten male mice aged 6-8 weeks were euthanized, ensuring complete death. The mice were then disinfected by immersion in 75% ethanol and transferred to a laminar flow hood for dissection. The skin and peritoneum were incised using sterile instruments, and the liver was completely removed and placed in an EP tube for preservation on ice.

[0150] 3. Inside the biosafety cabinet, transfer the liver from the EP tube to a 60mm sterile culture dish (culture dish a). Add 10mL of pre-chilled PBS buffer and gently rinse twice to thoroughly remove any hair and bloodstains adhering to the liver surface. Then transfer the cleaned liver to a new sterile culture dish (culture dish b). Spread a 100μm pore size filter membrane evenly on the tissue fragments, add 5mL of preheated 5% CM culture medium, and gently and thoroughly mechanically grind using the flat end of a syringe plunger to obtain a cell suspension.

[0151] 4. Filter the cell suspension through a 100μm cell sieve into a 15mL centrifuge tube to remove undigested connective tissue and cell clumps. Centrifuge the filtrate at 660×g at room temperature for 5 minutes, carefully discard the supernatant, and gently resuspend the cell pellet in 5mL of 5% CM medium. Then, slowly stack the cell suspension onto an equal volume of preheated Ficoll lymphocyte separation medium, taking care to maintain a clear interface between the two phases. Perform gradient centrifugation at 1800 r / min for 20 minutes (room temperature, speed 9 for ascending, speed 1 for descending). This step effectively separates cell populations of different densities.

[0152] 5. 15 minutes before the end of gradient centrifugation, remove the guinea pig complement reagent and CD90.2 (Thy-1.2) antibody from the refrigerator and place them on ice to thaw.

[0153] 6. After centrifugation, use a pipette to aspirate the white membrane layer at the Ficoll interface and transfer it to a new 15mL centrifuge tube. Add 5% CM culture medium to a final volume of 10mL, mix gently, and centrifuge at 660×g for 5 minutes to wash. Discard the supernatant, take a small amount of cell suspension and mix it with 0.4% trypan blue solution (1:1), and use a hemocytometer to count cells and detect cell viability.

[0154] 7. According to the experimental design, the cells were randomly divided into two groups (n=5 / group). The experimental data showed that the number of white blood cell layers extracted from the livers of 10 mice was as follows: Unpurified group ① 3.4×10 6 ②4.6×10 6 ③3.7×10 6 ④2.2×10 6 ⑤ 4.3×10 6 Purification group ⑥ 3.9×10 6 ⑦4.1×10 6 ⑧3.8×10 6 ⑨3.7×10 6 ⑩2.9×10 6 The unpurified group was directly taken at a rate of 1×10⁻⁶. 6 Cells were subjected to flow cytometry staining; the purified group underwent B-cell positive screening using the following formula, with the required amounts of each reagent calculated accordingly:

[0155] (1) 1×HBSS buffer volume (V1 mL) = Total cell number / (4×10) 7 );

[0156] (2) Complement volume (V2 mL) = V1 / 10;

[0157] (3) Thy-1.2 antibody volume (V3 μL) = V1 (numerical value) × 2;

[0158] 8. The required reagent volumes for each sample in the purification group were calculated as follows: ⑥ V1 = 0.098 mL, V2 = 0.010 mL, V3 = 0.196 μL ⑦ V1 = 0.1025 mL, V2 = 0.010 mL, V3 = 0.205 μL ⑧ V1 = 0.095 mL, V2 = 0.010 mL, V3 = 0.19 μL ⑨ V1 = 0.0925 mL, V2 = 0.009 mL, V3 = 0.185 μL ⑩ V1 = 0.0725 mL, V2 = 0.007 mL, V3 = 0.145 μL;

[0159] 9. Incubate the prepared cell-antibody-complement mixture in a 37°C water bath for 30 minutes, gently inverting and mixing every 10 minutes. After incubation, add 1×HBSS buffer to a final volume of 10 mL and centrifuge at 660×g for 5 minutes. At this point, T cells will be lysed by complement due to the binding of the Thy-1.2 antigen and antibody on their surface, while B cells and monocytes will precipitate at the bottom of the tube.

[0160] 10. After discarding the supernatant, resuspend the cell pellet in 5 mL of 5% CM and transfer the suspension to a T75 culture flask. Wash the centrifuge tube twice with 5 mL of culture medium, and combine all the liquids into the same culture flask to make a final volume of 15 mL. Incubate the culture flask in a 37°C, 5% CO2 incubator for 1 hour to allow the monocytes to adhere. Then collect the non-adherent cell suspension, centrifuge at 660×g for 5 minutes to obtain an enriched B cell population.

[0161] 11. Flow cytometry analysis used the following antibody combination: total B cell marker was Anti-mouse-B220-BV605; apoptosis detection used 7-AAD-Percp + Annexin V-FITC; B cell subset markers were CD23-PE + CD21-PE-Cy7. 1×10⁻⁶ samples were taken from each sample. 6 Cells were incubated with antibody suspension (50 μL) for 30 minutes in the dark, followed by washing twice with PBS and resuspending in 200 μL PBS. Data were acquired using a Longcyte C3140 flow cytometer and Modeflower software, and the results were analyzed using FlowJo v10.9.0 software. Specific results are shown below. Figure 6 As shown.

[0162] 12. Cell counting was performed using a cell counting chamber by adding lymphocyte suspensions obtained from density gradient centrifugation of the unpurified and purified groups. The mean number of unpurified white membrane cells was (3.64±0.934)×10⁻⁶. 6 The mean number of white membrane cells in the purified group was (3.68±0.460)×10⁻⁶. 6The mean values ​​of the two sets of data showed no significant difference, allowing for comparison during subsequent purification operations.

[0163] 13. B-cell yield determination: Total B-cell markers were detected by flow cytometry as described above. The results showed that the B-cell yield in the unpurified group was 10.56±0.891%, while the B-cell yield in the purified group was significantly increased to 43.2±1.722%. The difference between the two groups was statistically significant. Specific results are as follows: Figure 6 As shown in A.

[0164] 14. Apoptosis Rate and Viability Identification: The flow cytometry method used to detect apoptotic cells showed that the apoptosis rate in the unpurified group was 0.208±0.091%, while the apoptosis rate in the purified group significantly decreased to 0.035±0.029%. The difference between the two groups was statistically significant. Specific results are as follows: Figure 6 As shown in B.

[0165] 15. Identification of B-cell subset proportions: B-cell subset markers were detected by flow cytometry as described above. The results showed that the proportion of follicular B cells in the unpurified group was 66.68±3.833%, while that in the purified group was 75.78±4.272%; the proportion of B1 B cells in the unpurified group was 20.7±3.104%, while that in the purified group was 13.3±2.426%. See below for details. Figure 6 C. This indicates that after purification, the proportion of FOB cells increased and the proportion of B1 B cells decreased. Furthermore, the various B cell subsets in both the unpurified and purified groups fluctuated within the normal range and did not affect the overall B cell subset classification. This provides a high-purity and high-activity cellular basis for subsequent B cell function studies.

[0166] 16. Immunofluorescence Detection: Images of unpurified B cells and primary B cells prepared using a Nikon A1R laser confocal microscope were acquired. B cells were activated with free antigens, and changes resulting from antigen receptor (BCR) activation were detected. The images were processed and analyzed using NIS element software to assess whether the activation function of the primary B cells extracted using this method was altered. Image results are shown below. Figure 6 As shown in Figure D, the changes in the surface of B cells extracted and purified by this method over time are within the normal range, confirming that the integrity of the extracted cell membrane proteins and their signal transduction function are normal, indicating that the B cells extracted by this method can be used for subsequent immunological research.

[0167] Table 5

[0168]

[0169] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing B lymphocytes, characterized in that, Includes the following steps: S1: Obtain a single-cell suspension; S2: Centrifuge the single-cell suspension using a density gradient to obtain an interface layer cell rich in mononuclear cells; S3: The interface layer cells were lysed using specific antibodies against T cell surface antigens and specific complement, centrifuged, and the precipitate was collected; S4: Collect the cells obtained from the precipitate in S3, purify them by adhering to the wall, remove the non-adherent cells, and obtain the B lymphocytes.

2. The preparation method according to claim 1, characterized in that, The density gradient centrifugation described in S2 uses Ficoll lymphocyte separation medium.

3. The preparation method according to claim 2, characterized in that, The volume ratio of the Ficoll lymphocyte separation solution to the single-cell suspension is 1:

1.

4. The preparation method according to claim 2 or 3, characterized in that, The density of the Ficoll lymphocyte separation solution is 1.083~1.085 g / mL.

5. The preparation method according to any one of claims 1 to 4, characterized in that, The specific antibodies include: CD90.2 (Thy-1.2) antibody.

6. The preparation method according to any one of claims 1 to 5, characterized in that, The specific complement includes: guinea pig complement.

7. The preparation method according to any one of claims 1 to 6, characterized in that, The T cell lysis also includes: containing Ca 2+ Mg 2+ HBSS buffer.

8. The preparation method according to any one of claims 1 to 7, characterized in that, The time for the adhesion purification is 45-65 minutes.

9. The preparation method according to any one of claims 1 to 8, characterized in that, The single-cell suspension was derived from the liver or spleen.

10. B lymphocytes obtained by the preparation method according to any one of claims 1 to 9.

Images