Lymphatic tissue flow detection sample preserving fluid as well as preparation method and application thereof

Abstract

The invention provides a lymphoid tissue flow detection sample preservation solution as well as a preparation method and application thereof. The preservation solution comprises DNA enzyme I, trehalose, ellagic acid and an RPMI 1640 culture medium. DNase I, trehalose and ellagic acid are added on the basis of an RPMI 1640 culture medium, the problems of DNA adhesion, cell membrane damage and microbial contamination existing in a traditional preservation solution are solved, and the preservation quality of a lymphatic tissue sample and the accuracy of subsequent analysis are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological sample preservation, and in particular to a lymphatic tissue flow cytometry sample preservation solution, a preparation method and an application thereof. Background Art

[0002] In immunological research and clinical diagnosis, the quality of lymphoid tissue samples directly impacts the accuracy and reliability of subsequent high-precision assays such as flow cytometry and single-cell transcriptome sequencing. Traditional lymphoid tissue sample preservation solutions often rely on polysucrose density gradient centrifugation for cell separation. While this method can effectively reduce cell aggregation, it still has significant drawbacks in practical applications. For example, it is unable to degrade free DNA, resulting in the frequent appearance of a network structure formed by nucleic acids in the resulting single-cell suspension, which affects the cell yield (usually less than 75%) and limits the sensitivity of subsequent analysis.

[0003] In addition, the chelating agent EDTA, which is widely used in existing preservation solutions, has certain hidden dangers in maintaining cell activity. Excessive EDTA will over-chelate the divalent calcium and magnesium ions in the solution, resulting in an abnormal increase in cell membrane permeability, especially when conducting calcium ion-dependent tests (such as Annexin V apoptosis tests), which can easily lead to false negative results. At the same time, to prevent microbial contamination, traditional formulas often add antibiotics such as gentamicin, but their antibacterial spectrum is limited, and they only have a certain inhibitory effect on Gram-negative bacteria, while being almost ineffective against fungal contamination, further increasing the risk in the sample preservation process. Therefore, the present application aims to provide a new type of lymphatic tissue flow cytometry sample preservation solution, its preparation method and application, so as to improve the sample preservation quality and the accuracy of subsequent testing while completely abandoning EDTA and sodium azide. Summary of the Invention

[0004] In view of this, the present invention proposes a lymphatic tissue flow cytometry sample preservation solution, a preparation method, and an application thereof, which improve the sample preservation quality and subsequent detection accuracy.

[0005] The technical solution of the present invention is implemented as follows: In a first aspect, the present invention provides a lymphatic tissue flow cytometry sample preservation solution, wherein the preservation solution comprises DNase I, trehalose, ellagic acid and RPMI 1640 culture medium.

[0006] DNase I specifically cleaves DNA phosphodiester bonds, eliminating the nucleic acid network. This prevents free DNA from forming a network that could hinder the preparation of single-cell suspensions, thereby improving single-cell yield and cell viability. DNase I works together with ellagic acid to reduce the effects of oxidative stress on cell membranes, ensuring high cell viability during cell isolation. Furthermore, DNase I combined with trehalose can further protect cells from physical damage.

[0007] Trehalose regulates the glass transition temperature, inhibiting ice crystal formation and maintaining the integrity of the cell membrane's liquid crystal structure, thereby improving cell survival and reducing the risk of cell rupture. Trehalose, when used in conjunction with DNase I, not only ensures the presence of extracellular substances that would hinder the formation of single-cell suspensions, but also physically protects the cell membrane from damage. Furthermore, its high osmotic pressure helps maintain a balance between the intracellular and extracellular environments, enhancing overall preservation effectiveness.

[0008] Ellagic acid through Fe 2+ Chelation and activation of the Nrf2 pathway reduce the rate of mitochondrial ROS production, effectively inhibiting lipid peroxidation levels and protecting cells from oxidative damage. Ellagic acid exhibits a certain inhibitory effect on bacteria and fungi, inhibiting the activity of certain pathogen enzymes, thereby hindering their growth and reproduction. Ellagic acid works together with DNase I to not only address external issues (such as DNA adhesion) but also address the antioxidant needs within the cell. Together, these two factors promote the maintenance of cellular health. The presence of ellagic acid also reduces the loss of cell function due to oxidative stress.

[0009] RPMI 1640 medium provides essential metabolic support, such as glucose and glutamine, ensuring that cells receive essential nutrients to maintain basic life activities during storage. Serving as the foundation of the entire formula, RPMI 1640 medium provides an optimal physiological environment for the other active ingredients to function. Combined with the three aforementioned ingredients, it forms a comprehensive system that supports cell health.

[0010] Based on the above technical solution, preferably, taking 1 L of preservation solution as an example, the concentration of DNase I is 10-20 U / mL, the concentration of ellagic acid is 0.1-0.5 mM, the concentration of trehalose is 2%-5% w / v, and the rest is RPMI 1640 culture medium.

[0011] On the basis of the above technical solution, preferably, propolis extract is also included.

[0012] Propolis extract contains broad-spectrum antimicrobial compounds (flavonoids and phenolic acids) with significant antimicrobial and antioxidant properties, effectively controlling microbial contamination, particularly against fungi such as Candida albicans. While propolis extract and DNase I act differently, both enhance sample quality—the former addresses biochemical issues, while the latter targets potential microbial threats. Beyond their respective primary functions, propolis extract and trehalose complement each other in protecting cellular integrity and resisting external stress. Both propolis extract and ellagic acid possess antioxidant capacity, and the flavonoids in propolis can enhance the antioxidant network, further mitigating cellular damage caused by oxidative stress. While RPMI 1640 medium provides essential nutrients, the addition of propolis extract enhances the overall preservation medium's ability to withstand adverse factors, creating a more stable microenvironment conducive to cell survival.

[0013] Based on the above technical solution, preferably, the concentration of the propolis extract in the preservation solution is 0.1%~0.5% w / v.

[0014] On the basis of the above technical solution, preferably, the propolis extract is prepared by supercritical CO2 extraction.

[0015] Based on the above technical solution, preferably, the extraction pressure is 25~35 MPa, the temperature is 40~50°C, the CO2 flow rate is 15~25 L / h, and the extraction time is 2~4h.

[0016] In a second aspect, the present invention provides a method for preparing a sample preservation solution for lymphoid tissue flow cytometry, comprising the following steps: dissolving DNase I in RPMI 1640 medium pre-cooled at 4°C to prevent loss of enzyme activity due to trehalose coating; adding trehalose and stirring at a low speed (200 rpm) to prevent foaming; then, sequentially adding ellagic acid and propolis extract, stirring thoroughly, and filtering to obtain the preservation solution.

[0017] Based on the above technical solution, preferably, the pH value of the preservation solution is 7.2-7.4.

[0018] In a third aspect, the present invention provides use of a preservation solution in the preparation of a lymph node single cell suspension.

[0019] The lymphatic tissue flow cytometry sample preservation solution and its preparation method and application of the present invention have the following beneficial effects compared with the prior art: (1) DNase I solves the physical obstruction problem caused by free DNA outside the cell, while trehalose protects the integrity of the cell membrane from a physical perspective. The two work together to ensure high cell yield and activity. Ellagic acid mainly protects against oxidative stress inside the cell, while DNase I and trehalose solve the problems of external DNA adhesion and cell membrane stability respectively. The three work synergistically to ensure the optimization of the extracellular environment and maintain intracellular homeostasis. RPMI 1640 culture medium, as a basic nutrient supply source, provides a suitable environment for other active ingredients, allowing these ingredients to perform their respective functions under optimal conditions, thereby comprehensively improving the sample preservation effect. These four ingredients, through their unique mechanisms and synergistic effects, jointly solve the three major technical problems existing in traditional preservation solutions: DNA adhesion, cell membrane damage and microbial contamination, significantly improving the quality of lymphoid tissue sample preservation and the accuracy of subsequent analysis.

[0020] (2) DNase I prevents the formation of DNA network structures, while propolis prevents the release of secondary DNA caused by microbial proliferation through its antibacterial effect, jointly ensuring the quality of single-cell suspension. Propolis extract provides antioxidant support and synergizes with trehalose to enhance the cell's resistance to damage from both physical protection and chemical defense levels. Both propolis extract and ellagic acid are antioxidant components. The flavonoids in propolis can enhance the activation effect of the Nrf2 pathway, further reduce ROS and MDA levels, and enhance cell activity. On the basis of the nutritional support of RPMI 1640 culture medium, propolis extract enhances the antibacterial and antioxidant functions, allowing the preservation solution to maintain the physiological state of cells during long-term storage. DETAILED DESCRIPTION

[0021] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0022] The DNA enzyme I used in the present invention was purchased from Wuhan Punuo Life Science Technology Co., Ltd., PMI 1640 culture medium was purchased from Wuhan Punuo Life Science Technology Co., Ltd.; ellagic acid was purchased from Merck Biotechnology.

[0023] The present invention does not specifically limit the source of propolis extract; it can be purchased from a commercial vendor or prepared through a process. The preparation method of the propolis extract in the embodiments of the present invention is as follows: select high-quality propolis raw material, remove impurities, and crush to a 60-mesh particle size. Vacuum dry the extract at 50°C for 3 hours to reduce the moisture content to ≤5% before use. The dried propolis powder is placed in a supercritical extraction kettle at a loading volume of 70% of the kettle volume. Extraction parameters are set as follows: pressure 30 MPa, temperature 45°C, CO2 flow rate 20 L / h (per kilogram of raw material), and extraction time 4 hours. After extraction, the CO2 is separated to obtain a crude propolis extract rich in flavonoids, phenolic acids, and terpenes. The extract is then concentrated under reduced pressure to remove residual solvent, and freeze-dried to obtain the finished product.

[0024] Example 1 The lymphoid tissue flow cytometry sample preservation solution of this embodiment includes DNase I, trehalose, ellagic acid, and RPMI 1640 medium. Taking 1 L of preservation solution as an example, the concentration of DNase I is 15 U / mL, the concentration of ellagic acid is 0.3 mM, the concentration of trehalose is 4% w / v, and the remainder is RPMI 1640 medium.

[0025] The method for preparing a sample preservation solution for lymphoid tissue flow cytometry includes the following steps: dissolving DNase I in RPMI 1640 medium pre-chilled at 4°C to prevent loss of enzyme activity due to trehalose coating. Trehalose is added and stirred at a low speed (200 rpm) to prevent foaming. Ellagic acid is then added, stirred thoroughly, and the pH is adjusted to 7.4. The preservation solution is then filtered through a 0.22 μm PES membrane to obtain the preservation solution.

[0026] Example 2 The difference between Example 2 and Example 1 is that propolis extract is added, specifically: The lymphoid tissue flow cytometry sample preservation solution of this embodiment includes DNase I, trehalose, ellagic acid, and RPMI 1640 medium. Taking 1 L of preservation solution as an example, the concentration of DNase I is 15 U / mL, the concentration of ellagic acid is 0.3 mM, the concentration of trehalose is 4% w / v, the concentration of propolis extract is 0.4% w / v, and the remainder is RPMI 1640 medium.

[0027] The method for preparing a sample preservation solution for lymphoid tissue flow cytometry includes the following steps: dissolving DNase I in RPMI 1640 medium pre-cooled at 4°C to prevent loss of enzyme activity due to trehalose coating. Trehalose is added and stirred at a low speed (200 rpm) to prevent foaming. Ellagic acid and propolis extract are then added in sequence, stirred thoroughly, and the pH is adjusted to 7.4. The solution is then filtered through a 0.22 μm PES membrane to obtain the preservation solution.

[0028] Example 3 The lymphoid tissue flow cytometry sample preservation solution of this embodiment includes DNase I, trehalose, ellagic acid, propolis extract, and RPMI 1640 medium. Taking 1 L of preservation solution as an example, the concentration of DNase I is 10 U / mL, the concentration of ellagic acid is 0.2 mM, the concentration of trehalose is 2% w / v, the concentration of propolis extract is 0.2% w / v, and the remainder is RPMI 1640 medium.

[0029] The method for preparing a sample preservation solution for lymphoid tissue flow cytometry includes the following steps: dissolving DNase I in RPMI 1640 medium pre-cooled at 4°C to prevent loss of enzyme activity due to trehalose coating. Trehalose is added and stirred at a low speed (200 rpm) to prevent foaming. Ellagic acid and propolis extract are then added in sequence, stirred thoroughly, and the pH is adjusted to 7.2. The solution is then filtered through a 0.22 μm PES membrane to obtain the preservation solution.

[0030] Example 4 The lymphoid tissue flow cytometry sample preservation solution of this embodiment includes DNase I, trehalose, ellagic acid, propolis extract, and RPMI 1640 medium. Taking 1 L of preservation solution as an example, the concentration of DNase I is 20 U / mL, the concentration of ellagic acid is 0.5 mM, the concentration of trehalose is 5% w / v, the concentration of propolis extract is 0.5% w / v, and the remainder is RPMI 1640 medium.

[0031] The method for preparing a sample preservation solution for lymphoid tissue flow cytometry includes the following steps: dissolving DNase I in RPMI 1640 medium pre-cooled at 4°C to prevent loss of enzyme activity due to trehalose coating. Trehalose is added and stirred at a low speed (200 rpm) to prevent foaming. Ellagic acid and propolis extract are then added in sequence, stirred thoroughly, and the pH is adjusted to 7.4. The solution is then filtered through a 0.22 μm PES membrane to obtain the preservation solution.

[0032] Example 5 The lymphoid tissue flow cytometry sample preservation solution of this embodiment includes DNase I, trehalose, ellagic acid, propolis extract, and RPMI 1640 medium. Taking 1 L of preservation solution as an example, the concentration of DNase I is 12 U / mL, the concentration of ellagic acid is 0.1 mM, the concentration of trehalose is 3% w / v, the concentration of propolis extract is 0.1% w / v, and the remainder is RPMI 1640 medium.

[0033] The method for preparing a sample preservation solution for lymphoid tissue flow cytometry includes the following steps: dissolving DNase I in RPMI 1640 medium pre-cooled at 4°C to prevent loss of enzyme activity due to trehalose coating. Trehalose is added and stirred at a low speed (200 rpm) to prevent foaming. Ellagic acid and propolis extract are then added in sequence, stirred thoroughly, and the pH is adjusted to 7.3. The solution is then filtered through a 0.22 μm PES membrane to obtain the preservation solution.

[0034] Comparative Example 1 Compared with Example 1, Comparative Example 1 lacks ellagic acid, and the rest of the contents are the same.

[0035] Comparative Example 2 Compared with Example 1, in Comparative Example 2, the content of ellagic acid was 0.8 mM, and the rest of the contents were the same.

[0036] Comparative Example 3 Compared with Example 1, Comparative Example 3 lacks DNase I, and the rest of the contents are the same.

[0037] Comparative Example 4 Comparative Example 4 Compared with Example 1, the content of DNase I was 30 U / mL, and the rest of the contents were the same.

[0038] Comparative Example 5 Compared with Example 1, Comparative Example 5 lacks trehalose, and the rest of the contents are the same.

[0039] Comparative Example 6 Compared with Example 1, the content of trehalose in Comparative Example 6 is 6.5% (w / v), and the rest of the contents are the same.

[0040] Experiment 1 Preparation of mouse lymph node single cell suspension Operation process: Fresh mouse lymph node tissue was obtained, mechanically disrupted, and passed through a 100 μm cell sieve to prepare a single-cell suspension; the preservation solution of the present invention was added and stored at 4°C for 48 hours; cell death and viability were marked by PI, and the isolated lymphocytes were labeled with CD45 leukocyte common antigen, and cell activity and lymphocyte survival rate were detected by flow cytometry.

[0041] Microbial contamination rate detection: Take 1 mL of single-cell suspension and add it to a sterile test tube containing 9 mL of neutralizer (TPS buffer containing 0.5% sodium thiosulfate) to make a 1:10 dilution. Inoculate it into two sterile plates for incubation. Pour 15 mL of ordinary nutrient agar medium into each plate, mix well, and let solidify. Incubate at 36°C ± 1°C for 48 hours. The results are calculated according to the formula: Contamination rate (CFU / mL) = average colony count × dilution factor. If there is no sterile growth, report it as <5 CFU / mL.

[0042] The test results are shown in Table 1.

[0043] Table 1 Cell activity and contamination rate

[0044] EDTA tissue preservation solution is composed of chelating agent disodium ethylenediaminetetraacetic acid (EDTA-Na) + buffer (Tris-HCl) + preservative (sodium azide).

[0045] Formula (taking 1 L as an example): EDTA-Na2 stock solution concentration: 0.5 M, dosage: 20 mL, Tris-HCl (pH 8.0) stock solution concentration: 1 M, dosage: 10 mL, sodium azide (NaN3) stock solution concentration: 10% (w / v), dosage: 5 mL, dilute to 1 L with deionized water, and adjust to pH 8.0 In Comparative Example 1, the absence of ellagic acid increased the lipid peroxidation level (MDA) to 1.5 μM, doubled the ROS production rate, and decreased cell viability to 34.18%. In Comparative Example 2, the excessively high ellagic acid concentration had no significant toxicity, but it caused precipitation, so the concentration was limited to 0.1-0.5 mM. In Comparative Example 3, the absence of DNase I prevented the degradation of the nucleic acid network, resulting in a decrease in single-cell yield to 62.33% and a significant increase in cell aggregation to 28.4%. In Comparative Example 4, the excessively high DNase I concentration had no significant negative impact on the results, but it may have increased costs, so the concentration was limited to 10-20 U / mL. In Comparative Example 5, the absence of trehalose concentration led to insufficient osmotic pressure, increased membrane rupture rate, and a significant decrease in survival rate. In Comparative Example 6, the trehalose concentration was too high, exceeding the physiological range (345 mOsm / kg), resulting in a decrease in cell survival.

[0046] Experiment 2: Immunophenotype and gene expression stability experiment Test method: 1. Flow cytometry detection of immune cell proportion: After tissue preservation treatment, single cell suspension was prepared and incubated with CD3, CD19 and CD11c antibodies. CD3 was detected on a flow cytometer. + T cells, CD19 + B cells and CD11c + Dendritic cell ratio.

[0047] 2. qPCR analysis of inflammatory factors and apoptosis genes IL-6, TNF-α and Caspase-3 gene expression: Lyse cells to extract total RNA (1×10 6 cells), genomic DNA was digested with DNase I, cDNA was synthesized by reverse transcription, primers were designed, the reaction was performed at ×40 cycles, and specificity was verified by analysis.

[0048] 3. Long-term storage: After 72 hours of storage at 4°C, test cell viability. Trypan blue staining: Mix cells with 0.4% trypan blue (1:1) and count live cells. Proliferation capacity (CFSE staining): CFSE labeling, proliferation induction, culture at 37°C for 72 hours, flow cytometry: analyze the CFSE fluorescence dilution degree.

[0049] The test results are shown in Table 2.

[0050] Table 2 Immunophenotype and gene expression stability results

[0051] Detection of CD3 + T cells, CD19 + B cells and CD11c + The dendritic cell ratio is used to verify that the preservation solution effectively protects various immune cells and ensures that they do not undergo abnormal changes during the preservation process, thereby ensuring the authenticity and reliability of experimental data. This is crucial for subsequent analysis, as any deviation from the normal ratio may affect the interpretation of experimental results and may lead to erroneous conclusions.

[0052] The purpose of analyzing IL-6, TNF-α, and Caspase-3 gene expression is to assess the effects of the preservation solution on cell status and function, particularly the inflammatory response and apoptosis. A significant decrease in IL-6, TNF-α, and Caspase-3 mRNA expression (see Examples) indicates that not only has the risk of inflammatory response and apoptosis been reduced, but also that the cells' original physiological state has been better preserved. This is crucial for subsequent high-precision analysis such as flow cytometry and single-cell transcriptome sequencing.

[0053] The higher the CFSE staining ratio, the stronger the cell proliferation ability or the better the cell state; conversely, if the CFSE signal is lower than 50%, it may mean: cell death or damage, decreased proliferation ability.

[0054] CD3 of Examples 1-5 + T cells, CD19 + B cells and CD11c + The reduction rates of dendritic cell ratio and IL-6 / TNF-α / Caspase-3 mRNA (%) were higher than those of the control group, indicating that the preservation solution of the present application can effectively protect various immune cells, maintain the original physiological state of the cells, and ensure that they do not undergo abnormal changes during the preservation process.

[0055] This application ensures that 73.5% to 78.1% of the CFSE signal remains, indicating that the vast majority of cells have divided normally or are in a good physiological state; and the cell membrane is intact, the metabolism is active, and the proliferation capacity is stable. This shows that the preservation solution is non-toxic to cells and effectively prevents cell damage caused by oxidative stress, DNA adhesion, etc. The 27.4% CFSE signal remaining in the traditional EDTA preservation solution indicates that a large number of cells have failed to divide normally and may even die; the change in membrane permeability caused by EDTA may cause cell rupture or apoptosis, making it impossible for CFSE to be diluted normally.

[0056] Comparative Example 1 lacks ellagic acid and lacks antioxidant protection, which is manifested as a decrease in the proportion of immune cells; a significant increase in the expression of inflammatory factors and apoptosis genes; and a decrease in CFSE. Comparative Example 2 has an excessively high concentration of ellagic acid, which may cause precipitation or nonspecific toxicity; although it has an antioxidant effect, excessive amounts lead to impaired cell function, and the anti-inflammatory / anti-apoptotic effect is not as good as the optimal concentration. Comparative Example 3 Free DNA is not degraded, forming a network structure that hinders the preparation of single cell suspensions, which is manifested as: an increase in cell aggregation rate; CD 3+ The proportion of cells decreased significantly; apoptotic and inflammatory signals were enhanced; and CFSE was significantly reduced. In Comparative Example 4, the DNase I concentration was too high. Although it did not directly toxic the cells, it increased costs and did not provide further improvement. Compared with Example 1, it was slightly improved, but the increase was limited; the cost-effectiveness was not high; and the CFSE was still lower than that of Example 2. Comparative Example 5 lacked trehalose, resulting in loss of cell membrane stability and an increased risk of ice crystal formation. This was manifested as: an increased cell rupture rate, a decrease in the proportion of various immune cells, an aggravation of inflammatory and apoptotic responses, and a decrease in CFSE staining. Comparative Example 6 had an excessively high trehalose concentration, exceeding the physiological osmotic pressure range, resulting in cell dehydration or swelling. This was manifested as: impaired cell activity, weakened anti-inflammatory and anti-apoptotic effects, and decreased proliferation capacity (decreased CFSE).

[0057] Experiment 3 Biosafety Assessment The toxicity of the preservation solution to HEK293 cells was detected by CCK-8 assay. The specific method was as follows: HEK293 cells in the logarithmic growth phase were obtained, digested, centrifuged and collected, resuspended in serum-containing medium, and inoculated into CCK-8 assay plates at an adjusted density. The cells were pre-cultured in a CO2 incubator for 24 hours to allow the cells to fully adhere to the wall. The old medium was removed, and fresh medium containing gradient concentrations of the preservation solution was added to the experimental group, while the control group only received fresh medium. The cells were incubated for another 48 hours, and CCK-8 was added for color development. The OD value was detected by a microplate reader. Endotoxin content was determined using the Limulus amebocyte lysate (LAI) method: refer to the photometric method (turbidimetric / chromogenic method) in the Test Method for Bacterial Endotoxins (Chinese Pharmacopoeia). Steps: endotoxin standard was diluted in a gradient to cover the detection range of LAI. The test solution was mixed with LAI. A standard curve group and a negative control were set up. The absorbance was continuously monitored at 37°C using the dynamic turbidimetric method. The time required for the turbidity to rise to the threshold was recorded. The instrument automatically fitted the standard curve (endotoxin concentration vs. reaction time / rate) to calculate the endotoxin content of the test solution.

[0058] The preservation solution of Example 1 was used as the test sample, and the test results are shown in Table 3.

[0059] Table 3 Safety of preservation solution

[0060] As shown in Table 3, the biosafety of the preservation solution of the present application complies with ISO 10993-5 and has a low endotoxin content.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A sample storage solution for lymphatic tissue flow cytometry, characterized by: The preservation solution includes DNase I, trehalose, ellagic acid and RPMI 1640 culture medium.

2. The lymphatic tissue flow cytometry sample storage solution according to claim 1, characterized in that: Taking 1 L of preservation solution as an example, the concentration of the DNase I is 10-20 U / mL, the concentration of ellagic acid is 0.1-0.5 mM, the concentration of trehalose is 2%-5% w / v, and the rest is RPMI 1640 culture medium.

3. The lymphatic tissue flow cytometry sample storage solution according to claim 2, characterized in that: Also includes propolis extract.

4. The lymphatic tissue flow cytometry sample storage solution according to claim 3, characterized in that: The concentration of the propolis extract in the preservation solution is 0.1% to 0.5% w / v.

5. The lymphatic tissue flow cytometry sample storage solution according to claim 3, characterized in that: The propolis extract is prepared by supercritical CO2 extraction.

6. The lymphatic tissue flow cytometry sample storage solution according to claim 5, characterized in that: The extraction pressure is 25~35 MPa, the temperature is 40~50℃, the CO2 flow rate is 15~25 L / h, and the extraction time is 2~4h.

7. The method for preparing a lymphatic tissue flow cytometry sample preservation solution according to any one of claims 3 to 6, characterized in that: The following steps are involved: DNase I was dissolved in RPMI 1640 culture medium, and then trehalose, ellagic acid and propolis extract were added in sequence, stirred evenly and filtered to obtain a preservation solution.

8. The method for preparing a lymphatic tissue flow cytometry sample preservation solution according to claim 7, characterized in that: The pH value of the preservation solution is 7.2-7.

4.

9. Use of the preservation solution according to any one of claims 1 to 6 in the preparation of lymph node single cell suspension.

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