Solid tissue flow detection sample preserving fluid and preparation method thereof
By combining N-acetylcysteine, melatonin, lactobionic acid and HBSS buffer, combined with the use of ellagic acid, the problems of insufficient cell activity and antigen epitope stability in existing preservation solutions were solved, achieving efficient cell preservation and detection effects.
Patent Information
- Application Number
- CN202510823603.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-19
- Publication Date
- 2025-09-16
AI Technical Summary
Existing solid tissue flow cytometry sample preservation fluid has difficulty maintaining cell activity within 72 hours, with cell viability dropping below 80%. It also lacks effective antioxidant and anti-apoptotic components, affecting cell integrity and high-precision detection.
A combination of N-acetylcysteine, melatonin, lactobionic acid and HBSS buffer was used to scavenge ROS through antioxidant pathways, inhibit the ferroptosis pathway, maintain mitochondrial function, and add ellagic acid to inhibit cathepsin B activity, thereby improving cell survival and antigen epitope stability.
Significantly improve cell survival rate to over 95%, reduce nuclear fragmentation rate, maintain antigen epitope stability, inhibit microbial contamination, and ensure high-precision detection data quality.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of tissue preservation fluids, and in particular to a sample preservation fluid for flow cytometry detection of solid tissues and a preparation method thereof. Background Art
[0002] In biomedical research and clinical diagnosis, high-quality preservation of solid tissue samples (such as tumor, liver, brain tissue, etc.) is crucial for subsequent high-precision detection methods such as flow cytometry and single-cell transcriptome sequencing.
[0003] Currently, preservation solutions for flow cytometry samples of solid tissues (such as tumors, liver, and brain tissue) generally suffer from poor short-term preservation performance. Conventional preservation solutions struggle to maintain cell viability within 72 hours, primarily manifesting as a drop in cell viability below 80%. This is primarily due to oxidative stress, which leads to decreased mitochondrial membrane potential and elevated levels of lipid peroxidation products (MDA), which in turn impair cell function and even cell death. Furthermore, existing formulations lack effective antioxidant and anti-apoptotic components, making them incapable of meeting the cell integrity requirements for high-precision assays. Existing preservation solutions also exhibit significant deficiencies in protein stability and preservation. The lack of specific tissue protease inhibitors results in nuclear fragmentation rates exceeding 50%, severely impacting data quality from flow cytometry and single-cell transcriptome sequencing. Furthermore, to control microbial contamination, conventional formulations rely on high concentrations of sodium azide, a component that not only interferes with cellular metabolism and enzyme activity but can also affect the accuracy of subsequent molecular experiments. Therefore, a new preservation solution that balances cell viability and antigen epitope stability is urgently needed. Summary of the Invention
[0004] In view of this, the present invention proposes a sample preservation solution for flow cytometry of solid tissues that takes into account both cell activity maintenance and antigen epitope stability, and a preparation method thereof.
[0005] The technical solution of the present invention is implemented as follows: In a first aspect, the present invention provides a solid tissue flow cytometry sample preservation solution, wherein the preservation solution comprises N-acetylcysteine, melatonin, lactobionic acid and HBSS buffer.
[0006] N-acetylcysteine (NAC) directly neutralizes reactive oxygen species (ROS) in the cytoplasm by providing sulfhydryl groups (-SH), reduces the damage of oxidative stress to cells, protects mitochondrial membrane potential, and prevents the accumulation of lipid peroxidation products (MDA). 13 H 16 N2O2) targets and stabilizes mitochondrial membrane potential, inhibits proteins related to the ferroptosis pathway, and thus alleviates cell damage caused by iron-dependent lipid peroxidation. 12 H 22 O 12) has antibacterial effects and can chelate iron ions, reduce free radical generation, and inhibit ferroptosis; it also enhances the overall antioxidant defense system. HBSS buffer maintains osmotic pressure and Ca² + / Mg² + Homeostasis provides a stable environment for other active ingredients, supporting essential cellular functions. NAC, melatonin, and lactobionic acid work together to activate antioxidant pathways, scavenging ROS, reducing the accumulation of lipid peroxidation products (MDA), protecting mitochondrial function, and enhancing cell survival. Melatonin and lactobionic acid work together to inhibit ferroptosis, maintaining cellular health, improving cell survival, and stabilizing antigenic epitopes. All these ingredients, combined with the stabilizing environment provided by HBSS, work together to maintain high cell viability and antigenic epitope stability.
[0007] Based on the above technical solution, preferably, taking 1 L of preservation solution as an example, the concentration of N-acetylcysteine is 2-3 mM, the concentration of melatonin is 1.5-2 mM, the concentration of lactobionic acid is 0.5-1.5 mM, and the rest is HBSS buffer.
[0008] Based on the above technical solution, preferably, the molar ratio of lactobionic acid to N-acetylcysteine is 1:1.9~2.1.
[0009] On the basis of the above technical solution, preferably, ellagic acid is also included.
[0010] By chelating metal ions, ellagic acid can inhibit the activity of cathepsin B, reduce the rate of nuclear fragmentation, and improve the quality of single-cell suspensions. The metal ion chelation effect of ellagic acid can assist in the antioxidant process and reduce lipid peroxidation, which complements the antioxidant mechanisms of NAC, melatonin, and lactobionic acid. Ellagic acid and NAC both help reduce oxidative damage, and ellagic acid can further inhibit the activation of proteases caused by oxidative stress and protect antigenic structure. Ellagic acid and melatonin work together to stabilize mitochondrial membrane potential, and ellagic acid additionally provides inhibition of proteases released by lysosomal rupture, doubly safeguarding cell integrity and antigenic epitope stability. Ellagic acid and lactobionic acid can both chelate metal ions, and ellagic acid adds inhibition of specific proteases on this basis, strengthening protection against oxidative stress and cell damage.
[0011] NAC, melatonin, lactobionic acid, and HBSS buffer have established a foundational system for effective antioxidant, anti-ferroptosis, and cell viability maintenance. The addition of ellagic acid specifically addresses the protein degradation problem present in traditional preservation solutions, significantly reducing the nuclear fragmentation rate and enhancing the protection of surface antigens. Ellagic acid not only possesses powerful antioxidant properties on its own but also forms a multi-layered synergistic effect with other ingredients, making the entire preservation solution formula even more effective in enhancing cell viability, stabilizing antigenic epitopes, and inhibiting microbial contamination.
[0012] Based on the above technical solution, preferably, the concentration of ellagic acid in the preservation solution is 0.2% to 0.4% w / v.
[0013] Based on the above technical solution, preferably, the pH value of the preservation solution is 7.2±0.1.
[0014] In a second aspect, the present invention provides a method for preparing a sample preservation solution for flow cytometry detection of solid tissue, comprising the following steps: S1, lactobionic acid and N-acetylcysteine were dissolved in HBSS buffer and stirred in the dark; S2, adding melatonin and ellagic acid to the mixed solution of step S1 in sequence, stirring evenly, adjusting the pH to 7.2±0.1 with 0.1 M NaOH, and then filtering to obtain a preservation solution.
[0015] In a third aspect, the present invention provides the use of a preservation solution in the preparation of a single cell suspension of solid tissue.
[0016] Based on the above technical solution, preferably, the solid tissue is one of liver and brain tissue.
[0017] The present invention provides a sample preservation solution for flow cytometry of solid tissues and a preparation method thereof, which has the following advantages over the prior art: (1) NAC, melatonin, and lactobionic acid work together to activate antioxidant pathways, scavenging ROS, reducing the accumulation of lipid peroxidation products (MDA), protecting mitochondrial function, and improving cell survival. Melatonin and lactobionic acid work together to inhibit the ferroptosis pathway, maintaining cell health, and improving cell survival and epitope stability. All of these components, along with the stable environment provided by HBSS, work together to maintain high cell viability and epitope stability.
[0018] (2) NAC, melatonin, lactobionic acid, and HBSS buffer solution form an effective basic system for antioxidant, anti-ferroptosis, and maintaining cell viability. By adding ellagic acid, the protein degradation problem existing in traditional preservation solutions is specifically targeted, the nuclear fragmentation rate is significantly reduced, and the protection of surface antigens is enhanced. Ellagic acid not only has a strong antioxidant capacity itself, but also forms a multi-level synergistic effect with other ingredients, making the entire preservation solution formula more excellent in improving cell activity, antigen epitope stability, and inhibiting microbial contamination. DETAILED DESCRIPTION
[0019] 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.
[0020] Melatonin used in this application was purchased from Merck Biotechnology; ellagic acid was purchased from Merck Biotechnology; and HBSS buffer was purchased from Wuhan Punosai Life Science Technology Co., Ltd.
[0021] Example 1 The preservation solution of this embodiment includes N-acetylcysteine, melatonin, lactobionic acid and HBSS buffer. Taking 1L of preservation solution as an example, the concentration of N-acetylcysteine is 2mM, the concentration of melatonin is 1.8mM, the concentration of lactobionic acid is 1mM, and the rest is HBSS buffer.
[0022] A method for preparing a sample preservation solution for flow cytometry detection of solid tissues comprises the following steps: S1, lactobionic acid and N-acetylcysteine were dissolved in HBSS buffer and stirred in the dark; S2, adding melatonin to the mixed solution of step S1, stirring evenly, adjusting the pH to 7.2, and then filtering with a 0.22 μm filter membrane to obtain a preservation solution.
[0023] Example 2
[0024] Example 2 adds ellagic acid on the basis of Example 1, specifically: The preservation solution of this embodiment includes N-acetylcysteine, melatonin, lactobionic acid, ellagic acid, and HBSS buffer. Taking 1 L of preservation solution as an example, the concentration of N-acetylcysteine is 2.4 mM, the concentration of melatonin is 1.8 mM, the concentration of lactobionic acid is 1.2 mM, the concentration of ellagic acid is 0.3% w / v, and the remainder is HBSS buffer.
[0025] A method for preparing a sample preservation solution for flow cytometry detection of solid tissues comprises the following steps: S1, lactobionic acid and N-acetylcysteine were dissolved in HBSS buffer and stirred in the dark; S2. Add melatonin and ellagic acid to the mixed solution of step S1, stir evenly, adjust the pH to 7.2, and then filter through a 0.22 μm filter membrane to obtain a preservation solution.
[0026] Example 3
[0027] The preservation solution of this embodiment includes N-acetylcysteine, melatonin, lactobionic acid, ellagic acid, and HBSS buffer. Taking 1 L of preservation solution as an example, the concentration of N-acetylcysteine is 2.85 mM, the concentration of melatonin is 1.7 mM, the concentration of lactobionic acid is 1.5 mM, the concentration of ellagic acid is 0.4% w / v, and the remainder is HBSS buffer.
[0028] A method for preparing a sample preservation solution for flow cytometry detection of solid tissues comprises the following steps: S1, lactobionic acid and N-acetylcysteine were dissolved in HBSS buffer and stirred in the dark; S2. Add melatonin and ellagic acid to the mixed solution of step S1, stir evenly, adjust the pH to 7.2, and then filter through a 0.22 μm filter membrane to obtain a preservation solution.
[0029] Example 4
[0030] The preservation solution of this embodiment includes N-acetylcysteine, melatonin, lactobionic acid, ellagic acid, and HBSS buffer. Taking 1 L of preservation solution as an example, the concentration of N-acetylcysteine is 3 mM, the concentration of melatonin is 1.5 mM, the concentration of lactobionic acid is 1.43 mM, the concentration of ellagic acid is 0.35% w / v, and the remainder is HBSS buffer.
[0031] A method for preparing a sample preservation solution for flow cytometry detection of solid tissues comprises the following steps: S1, lactobionic acid and N-acetylcysteine were dissolved in HBSS buffer and stirred in the dark; S2, adding melatonin and ellagic acid to the mixed solution of step S1, stirring evenly, adjusting the pH to 7.3, and then filtering with a 0.22 μm filter membrane to obtain a preservation solution.
[0032] Example 5
[0033] The preservation solution of this embodiment includes N-acetylcysteine, melatonin, lactobionic acid, ellagic acid, and HBSS buffer. Taking 1 L of preservation solution as an example, the concentration of N-acetylcysteine is 2.5 mM, the concentration of melatonin is 2 mM, the concentration of lactobionic acid is 0.5 mM, the concentration of ellagic acid is 0.2% w / v, and the remainder is HBSS buffer.
[0034] A method for preparing a sample preservation solution for flow cytometry detection of solid tissues comprises the following steps: S1, lactobionic acid and N-acetylcysteine were dissolved in HBSS buffer and stirred in the dark; S2. Add melatonin and ellagic acid to the mixed solution of step S1, stir evenly, adjust the pH to 7.1, and then filter through a 0.22 μm filter membrane to obtain a preservation solution.
[0035] Comparative Example 1 Compared with Example 1, Comparative Example 1 lacks melatonin, and the rest of the contents are the same.
[0036] Comparative Example 2 Compared with Example 1, in Comparative Example 2, the amount of melatonin exceeded the specified range, specifically 4 mM, and the rest of the contents were the same.
[0037] Comparative Example 3 Compared with Example 1, Comparative Example 3 lacks lactobionic acid, and the rest of the contents are the same.
[0038] Comparative Example 4 In Comparative Example 4, compared with Example 1, the amount of lactobionic acid used exceeded the specified range, specifically 2 mM, and the rest of the contents were the same.
[0039] Comparative Example 5 Comparative Example 5 is compared with Example 1, except that N-acetylcysteine is lacking, and the rest of the contents are the same.
[0040] Comparative Example 6 Compared with Example 2, in Comparative Example 6, the amount of ellagic acid exceeded the specified range, specifically 0.8% w / v, and the rest of the contents were the same.
[0041] Experiment 1: Preservation of mouse liver tissue Operation process: Fresh mouse liver tissue was taken and placed in the preservation solution prepared in the examples of the present invention and the comparative example, and stored in the dark at 4°C for 48 hours; the tissue was taken out and ground to prepare a single-cell suspension, and the cell life and death was marked by PI, and the epithelial cells were marked by EpCAM epithelial cell adhesion molecule antibody. Flow cytometry was used to detect the life and death of PI staining and EpCAM protein. The results are shown in Table 1.
[0042] Experiment 2: Enzyme activity inhibition Test method: Colorimetric determination of residual cathepsin B activity: The sample concentration was adjusted to 50 μg / 5 μL to avoid inhibition by high-concentration substrates or insufficient signal at low concentrations. A blank control was set to deduct background absorbance. A cathepsin B inhibitor (Cathepsin purchased from MCEchemexpress) was added. The samples were incubated at pH 5.5 and 37°C for 90 minutes. The samples were detected by microplate reader to verify whether the enzyme activity was completely inhibited. The accuracy of the calibration curve was verified by using activity standards.
[0043] The residual activity of cathepsin B was determined by colorimetry. The results are shown in Table 1.
[0044] Table 1 Cell viability, contamination rate and enzyme activity
[0045] EDTA tissue preservation solution is composed of chelating agent disodium ethylenediaminetetraacetic acid (EDTA-Na) + buffer (Tris-HCl) + preservative (sodium azide).
[0046] 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 A high cathepsin B inhibition rate (%) indicates the ability to protect cell structural integrity and effectively prevent cathepsin B from damaging internal cellular structures, particularly nuclear fragmentation caused by nuclear membrane rupture and chromatin leakage. It also reduces programmed cell death (such as apoptosis or necrosis) mediated by cathepsin B, thereby maintaining higher cell survival rates. For the preparation of high-quality single-cell suspensions, a high inhibition rate helps maintain cell morphological integrity and antigen epitope stability, resulting in more accurate and reliable experimental data. This is particularly important for techniques such as flow cytometry and single-cell transcriptome sequencing.
[0047] As shown in Table 1, the preservation solution of Example 1 significantly improved cell viability (>95%) and antigen epitope stability (cathepsin B inhibition rate >91%) in flow cytometry. The addition of ellagic acid in Example 2 resulted in higher cell viability, more stable antigen epitopes, and improved antigen structural integrity.
[0048] Comparative Example 1 lacks melatonin, resulting in a decrease in mitochondrial membrane potential, aggravation of oxidative stress, partial degradation of antigens, and then reduction in cell activity, partial degradation of antigens, and obvious antigen denaturation, indicating that melatonin is an important component for maintaining high cell activity and antigen stability. Comparative Example 2 melatonin content is excessive, and toxicity may be produced, or interfere with other components, affecting metabolic balance and antigen expression, and then reducing cell activity antigen expression. Comparative Example 3 lacks lactobionic acid, loses antibacterial and anti-ferroptosis dual functions, resulting in a decrease in cell activity, with antigen slightly degraded, and oxidative stress increased, indicating that lactobionic acid is a key component for controlling microbial contamination and auxiliary antioxidant. When Comparative Example 4 lactobionic acid is excessive, it will break the synergistic balance with other components, but is not conducive to overall performance. Comparative Example 5 lacks NAC, loses major antioxidant capacity, and ROS accumulation is serious, and cell activity and cathepsin B inhibition rate are reduced, indicating that NAC is the core component for removing ROS, and lack will significantly reduce preservation quality. Comparative Example 6 ellagic acid is excessive, and cell activity is not improved. Although ellagic acid is beneficial, its effect has a platform effect, and excessive use has a low cost performance.
[0049] 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 preservation solution for flow cytometry of solid tissues, characterized by: The preservation solution includes N-acetylcysteine, melatonin, lactobionic acid and HBSS buffer.
2. The solid tissue flow cytometry sample preservation solution according to claim 1, characterized in that: Taking 1 L of preservation solution as an example, the concentration of N-acetylcysteine is 2-3 mM, the concentration of melatonin is 1.5-2 mM, the concentration of lactobionic acid is 0.5-1.5 mM, and the rest is HBSS buffer.
3. The solid tissue flow cytometry sample preservation solution according to claim 1, characterized in that: The molar ratio of lactobionic acid to N-acetylcysteine is 1:1.9-2.
1.
4. The solid tissue flow cytometry sample preservation solution according to claim 2, characterized in that: Also includes ellagic acid.
5. The solid tissue flow cytometry sample preservation solution according to claim 4, characterized in that: The concentration of ellagic acid in the preservation solution is 0.2% to 0.4% w / v.
6. The solid tissue flow cytometry sample preservation solution according to claim 1, characterized in that: The pH value of the preservation solution is 7.2±0.
1.
7. The method for preparing a sample preservation solution for flow cytometry of solid tissue according to any one of claims 3 to 6, characterized in that: The following steps are involved: S1, lactobionic acid and N-acetylcysteine were dissolved in HBSS buffer and stirred in the dark; S2, adding melatonin and ellagic acid to the mixed solution of step S1 in sequence, stirring evenly, adjusting the pH to 7.2±0.1, and then filtering to obtain a preservation solution.
8. Use of the preservation solution according to any one of claims 1 to 6 in the preparation of single-cell suspensions of solid tissues.
9. The use according to claim 8, characterized in that: The solid tissue is one of liver and brain tissue.