Kit for integrally detecting senescent cells and apoptotic cells as well as detection method and application of kit

Abstract

The invention provides a kit for integrally detecting aging cells and apoptotic cells as well as a detection method and application of the kit. The kit comprises an aging inducer, an apoptosis inducer, an integrated buffer solution, a beta-galactosidase specific fluorescent substrate and an apoptotic cell fluorescent staining solution. The kit disclosed by the invention contains an integrated buffer solution capable of fully meeting the detection requirements of senescence and apoptosis cell populations and transition cell populations, so that the experimental steps can be simplified to the greatest extent, and the experimental cost is saved. Results provided by the kit are visual, data are accurate, the detection accuracy and sensitivity are remarkably improved, and a powerful tool is provided for integrally analyzing and detecting dynamic evolution of the aging process and the apoptosis process through a streaming technology.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell biology and molecular biology, and in particular relates to a kit for integrated detection of senescent cells and apoptotic cells, and a detection method and application thereof. Background Art

[0002] With the acceleration of the global population aging trend and changes in lifestyle, cancer has become a major public health issue that seriously threatens human health and quality of life, and its global burden is becoming increasingly heavy. Faced with this challenge, the medical community is actively responding, constantly exploring and optimizing tumor treatment strategies, and adopting multi-dimensional treatment measures from traditional surgical treatment, radiotherapy, chemotherapy to cutting-edge targeted therapy and immunotherapy. Among them, the mechanism of promoting cancer cell apoptosis and aging is the key to anti-tumor therapy, and its research and application have attracted much attention.

[0003] Cell senescence, as a natural biological defense mechanism, has a profound impact on the development and treatment of cancer. Senescent tumor cells fall into an irreversible state of growth arrest, which greatly limits their proliferation potential, thereby effectively curbing the growth of tumors. At the same time, senescent cells actively recruit immune cells by secreting a series of biologically active molecules such as cytokines and chemokines, forming an immune clearance network against senescent cells and their surrounding pre-malignant tumor cells, further curbing the progression of tumor deterioration.

[0004] Apoptosis, as a highly conservative form of programmed cell death, ensures that damaged or abnormal cells in the body can be cleared in a timely manner, thus curbing the occurrence of tumors at the source. Promoting tumor cell apoptosis is crucial for tumor treatment. In tumor treatment, chemotherapy and radiotherapy induce DNA damage, activate related pathways, trigger a cascade reaction of cell aging and apoptosis, and achieve anti-tumor effects. In particular, chemotherapy drugs represented by doxorubicin, etoposide, and camptothecin, as well as platinum drugs such as cisplatin, carboplatin, and oxaliplatin, cause DNA damage through different mechanisms, activate key molecules such as p53, promote tumor cell aging and apoptosis, and show strong anti-tumor effects.

[0005] However, in current clinical practice, there are significant limitations in monitoring tumor cell senescence and apoptosis. Traditional methods often adopt a separate detection strategy, that is, evaluating the degree of cell senescence or cell apoptosis separately, which makes it difficult to fully capture the dynamic evolution of these two key processes of tumor cells after patients receive treatment, thereby limiting the accurate evaluation of drug efficacy. This situation urgently requires the development of an integrated kit that can simultaneously detect cell senescence and apoptosis to achieve a more comprehensive and in-depth insight into the treatment effect.

[0006] In summary, the development of an efficient kit that integrates cell senescence and apoptosis detection functions is an important innovation in the field of tumor treatment. It will profoundly affect the formulation and implementation of future tumor treatment strategies and provide cancer patients with more accurate, safe and effective treatment options. Summary of the invention

[0007] In view of this, the present invention aims to overcome the defects in the prior art and proposes a kit for detecting senescent cells and apoptotic cells in an integrated manner and a detection method and application thereof.

[0008] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0009] In a first aspect, the present invention provides an integrated kit for detecting senescent cells and apoptotic cells, comprising a senescence inducer, an apoptosis inducer, an integrated buffer, a β-galactosidase-specific fluorescent substrate, and an apoptotic cell fluorescent staining solution;

[0010] The integrated buffer comprises a nonionic amphoteric buffer, sodium chloride, calcium chloride, plant-derived recombinant human serum albumin, and erythritol. The nonionic amphoteric buffer is used at a concentration of 8-18 mM, the sodium chloride is used at a concentration of 127-154 mM, the calcium chloride is used at a concentration of 2.00-2.46 mM, the plant-derived recombinant human serum albumin is used at a concentration of 0.5-5.0 mg / ml, and the erythritol is used at a concentration of 0.35-0.83 mM.

[0011] Preferably, the nonionic amphoteric buffer is selected from one of 4-hydroxyethylpiperazineethanesulfonic acid, 2-morpholineethanesulfonic acid, and 3-(N-morpholino)propanesulfonic acid.

[0012] In some embodiments, the nonionic amphoteric buffer is 4-hydroxyethylpiperazineethanesulfonic acid, and the concentration of the 4-hydroxyethylpiperazineethanesulfonic acid is 10 mM.

[0013] In some embodiments, the sodium chloride is used at a concentration of 150 mM.

[0014] In some embodiments, the calcium chloride is used at a concentration of 2.46 mM.

[0015] In some embodiments, the plant-derived recombinant human serum albumin is used at a concentration of 1 mg / ml.

[0016] In some embodiments, the erythritol is used at a concentration of 0.56 mM.

[0017] Preferably, the pH value of the integrated buffer is 5.8-6.4.

[0018] In some embodiments, the pH of the integrated buffer is 6.0.

[0019] Preferably, the senescence inducing agent is doxorubicin.

[0020] In some embodiments, the senescence-inducing agent is subjected to senescence-inducing conditions at a concentration of 50-200 nM for 24-96 h.

[0021] In some embodiments, the senescence inducing condition is treatment at a concentration of 100 nM for 48 hours.

[0022] Preferably, the apoptosis-inducing agent is osimertinib.

[0023] In some embodiments, the apoptosis inducing condition of the apoptosis inducing agent is treatment at a concentration of 2.5-12.5 μM for 24-96 hours.

[0024] In some embodiments, the apoptosis inducing condition is treatment at a concentration of 10 μM for 48 hours.

[0025] In some embodiments, the integrated buffer is in (2-10)× concentrated form, for example, in 5× concentrated form, its components include 50 mM HEPES, 750 mM NaCl, 12.3 mM CaCl2, 2.5 mg / ml OsrHSA, 1.78 mM erythritol, and the pH value is precisely controlled to 6.0.

[0026] In some embodiments, the β-galactosidase-specific fluorescent substrate is C12FDG, which is used as an indicator for detecting senescent cells; the apoptotic cell staining solution is Annexin VAlexa Fluor 647, which is used as an indicator for detecting apoptotic cells.

[0027] In a second aspect, the present invention also provides a method for integrated detection of senescent cells and apoptotic cells using the above kit, comprising the following steps:

[0028] S1: Treat cell samples with senescence inducers and apoptosis inducers;

[0029] S2: Collect the cell supernatant and transfer it to a centrifuge tube, wash, and collect the washing solution and store it in a centrifuge tube;

[0030] Optionally, S3: add Bafilomycin A1 to pre-warmed fresh culture medium and incubate;

[0031] S3: discard the old culture medium in the cell sample, add the integrated buffer supplemented with β-galactosidase-specific fluorescent substrate, and add fetal bovine serum for incubation;

[0032] S4: After digesting the cells, the cell suspension is transferred to the centrifuge tube storing the supernatant collected in step S2, centrifuged, the supernatant is discarded, the cells are washed, centrifuged and the supernatant is discarded;

[0033] S5: Resuspend the cells in the integrated buffer to obtain a cell suspension and adjust the cell concentration to 1-4×10 6 Pieces / mL;

[0034] S6: Take the cell suspension prepared in step S5, add apoptotic cell fluorescent staining solution, mix well by pipetting, and incubate at room temperature in the dark;

[0035] S7: Add the integrated buffer to the cell sample for rehydration, mix the cell sample, filter it, detect it using a flow cytometer, and collect the fluorescence signal of the cell sample.

[0036] In a third aspect, the present invention also provides a use of the above-mentioned kit in the preparation of a combined drug for treating and / or preventing tumors.

[0037] In a fourth aspect, the present invention also provides a use of the above-mentioned kit in evaluating the safety-inducing concentration of a drug.

[0038] The cell state after drug treatment is very fragile and is easily disturbed by mechanical operation or external environmental stimulation during the detection process. These interference factors may induce or accelerate the cells to enter the state of aging and apoptosis, which has a serious impact on the accuracy of the test results. Therefore, protecting the stability of aging-related β-galactosidase (SA-β-Gal) and maintaining the integrity of the cell membrane (especially phosphatidylserine, as the binding site of the apoptosis detection molecule Annexin V) have become the core of the entire detection process. Among them, non-ionic amphoteric buffers can maintain the acid-base balance of the solution and ensure that the cells are in a stable pH environment during the detection process. It works synergistically with NaCl and CaCl2, in which NaCl is responsible for maintaining the normal osmotic pressure of the cell, and CaCl2, as a calcium ion donor, not only participates in the regulation of various cell signal transduction pathways, but also stabilizes the binding of apoptosis staining solution to phosphatidylserine on the apoptotic cell membrane. The above components jointly construct a cell environment that has both suitable osmotic pressure and appropriate ionic strength, which is conducive to indicator detection.

[0039] The pH value of the integrated buffer of the present invention is optimized to 6.0 to meet the requirements of SA-β-Gal activity detection. This weakly acidic environment can stimulate the activity of β-galactosidase in senescent cells, thereby improving the accuracy of detection.

[0040] The OsrHSA in the integrated buffer of the present invention is a recombinant human serum albumin derived from plants, which has the advantages of high purity, high stability and no impurities. It can be used as a protein stabilizer to effectively prevent enzymes and proteins from decomposing under adverse conditions such as acidic environment and high surface tension, further protecting SA-β-Gal, a key indicator of aging detection, and can be adsorbed on the solid phase surface to reduce nonspecific protein-protein interactions and improve detection specificity.

[0041] The erythritol in the integrated buffer of the present invention has a strong antioxidant property, which can protect the biological macromolecules in the solution and prevent them from being degraded due to factors such as oxidation and light. At the same time, it protects the cell membrane from oxidative stress damage and ensures the integrity of key molecules such as phosphatidylserine, thereby ensuring the accuracy of aging and apoptosis detection.

[0042] The experimental data of the present invention show that OsrHSA and erythritol have a synergistic effect. The synergistic effect of OsrHSA and erythritol is to further stabilize the integrity of the fluorescent dye, the key aging indicator β-galactosidase and the cell membrane. The combination of the two has a better effect.

[0043] The synergistic combination of the above innovatively designed buffer components not only optimizes the performance of fluorescent dyes and protects the activity of aging-related β-galactosidase, but also meets the conditions for detecting various cell populations and provides the cellular microenvironment required for detection, thereby significantly improving the sensitivity and accuracy of detection.

[0044] Compared with the prior art, the present invention has the following advantages:

[0045] The kit of the present invention contains an integrated buffer solution that can fully meet the detection requirements of two types of cell populations, aging and apoptosis, and transitional cell populations, which can simplify the experimental steps and save experimental costs to the greatest extent. The results are intuitive and the data are accurate, which improves the accuracy and sensitivity of the detection, and provides a powerful tool for the integrated analysis and detection of the dynamic evolution of the two processes of aging and apoptosis through flow cytometry. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a flow cytometry result diagram of the control group, aging-induced group, and apoptosis-induced group in Example 1;

[0047] Figure 2 is a flow cytometry result diagram of the control group, the single drug treatment group and the combined drug treatment group in Example 2;

[0048] Figure 3 yes Figure 2 Statistical diagram of the distribution ratio of each group of cells;

[0049] Figure 4This is a graph showing the detection results of the kit of the present invention detecting a specific concentration of doxorubicin at different treatment times;

[0050] Figure 5 is the proportion of senescent cells at different treatment times with a specific concentration of doxorubicin detected by SA-β-Gal chemical staining;

[0051] Figure 6 It is a comparison chart of the effects of the buffer components in the kit of the present invention;

[0052] Figure 7 The results of aging indexes detected by the kit of the present invention and the traditional aging reagent, and the simple combination of the traditional aging reagent and the apoptosis kit were compared;

[0053] Figure 8 The results of apoptosis indexes detected by integrated testing of the kit of the present invention and the traditional apoptosis kit, and the simple combination of the traditional aging reagent and the apoptosis kit were compared. DETAILED DESCRIPTION

[0054] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0055] In the present invention, "HEPES" refers to 4-hydroxyethylpiperazineethanesulfonic acid. "MES" refers to 2-morpholinoethanesulfonic acid. "MOPS" refers to 3-(N-morpholino)propanesulfonic acid. "OsrHSA" refers to plant-derived recombinant human serum albumin.

[0056] The present invention will be described in detail below with reference to the embodiments.

[0057] Example 1

[0058] The specific composition of the integrated kit for detecting senescent cells and apoptotic cells used in this example is shown in Table 1.

[0059] Table 1 Specific components of the kit

[0060] Component name Specifications (100T) Senescence Inducer: Doxorubicin 1mM 50μL Apoptosis inducer: Osimertinib 10mM 200μL 5× All-in-one Buffer 10mL Senescent cell stain: C12FDG 5mg dry powder Apoptotic cell staining solution: Annexin V Alexa Fluor 647 500μL

[0061] The composition of the 5× integrated buffer in Table 1 includes 50 mM HEPES, 750 mM NaCl, 12.3 mM CaCl2, 2.5 mg / ml OsrHSA, 1.78 mM erythritol, and the pH value is adjusted to 6.0.

[0062] Three groups of lung adenocarcinoma cell samples were prepared. The first group of cells was treated with the senescence inducer in the kit: 75nM doxorubicin for 36h; the second group of cells was treated with the apoptosis inducer: 5μM osimertinib for 12h; the third group of cells was used as the control group, and only the co-solvent DMSO was added. After the treatment, the status of the cell samples was detected using the kit of the present invention. The specific steps are as follows:

[0063] (1) Collect the cell supernatant and transfer it to a centrifuge tube;

[0064] (2) Wash twice with PBS, collect the washing solution in a centrifuge tube, and store at 4°C;

[0065] (3) Prepare a culture medium containing Bafilomycin A1. Add 1 mM Bafilomycin A1 (MCE, Catalog No. HY-100558) to the preheated fresh culture medium to a final concentration of 100 nM. Incubate at 37°C, 5% CO2 for 1 h.

[0066] (4) SA-β-Gal substrate enzymatic hydrolysis: discard the old culture medium, add C12FDG to the integrated buffer to a final concentration of 33 μM, and add 10% FBS to maintain cell nutrition. Incubate at 37°C, 5% CO2 for 2 h.

[0067] (5) Digest the cells with EDTA-free trypsin, centrifuge at 300 g, 4°C for 5 min, and collect the cells;

[0068] (6) Wash the cells twice with 4°C precooled PBS, centrifuge at 300g for 5 min each time, and discard the supernatant;

[0069] (7) Prepare 1× integrated buffer, resuspend the cells, and adjust the concentration to 1×10 6 cells / mL;

[0070] (8) Take 100 μL of the cell suspension prepared in step (7), add 5 μL of apoptotic cell fluorescent staining solution Annexin V-Alexa Fluor 647, mix well by pipetting, and incubate at room temperature in the dark for 5 min;

[0071] (9) Add 400 μL of 1× integrated buffer to each tube of sample;

[0072] (10) The samples were mixed, filtered, and detected by flow cytometry.

[0073] Test results and analysis:

[0074] The results are as follows Figure 1As shown, after the inducer contained in this kit is used to successfully induce senescence and apoptosis of cancer cells, the comprehensive state of senescence and apoptosis of cancer cells can be accurately and effectively detected by flow cytometry technology.

[0075] Example 2

[0076] The specific composition of the integrated kit for detecting senescent cells and apoptotic cells used in this example is shown in Table 1. The components of the 5× integrated buffer in Table 1 include 40 mM MES, 635 mM NaCl, 10 mM CaCl2, 2.5 mg / ml OsrHSA, 1.75 mM erythritol, and the pH value is adjusted to 6.0.

[0077] Lung adenocarcinoma cell PC-9 was used as a cell sample and divided into a control group, a single drug treatment group, and a combined drug treatment group. In the single drug treatment group, 100nM doxorubicin was used to induce cell senescence for 48 hours, and 10μM osimertinib was used for 24 hours and 48 hours to trigger cell apoptosis; the combined drug treatment group was set to first be treated with 100nM doxorubicin for 48 hours, and then combined with 10μM osimertinib for 24 hours and 48 hours. By comparing the cell responses of each group, the cell biological effects of the drugs alone and in combination were deeply analyzed. The specific steps are as follows:

[0078] (1) Collect the cell supernatant and transfer it to a centrifuge tube;

[0079] (2) Wash twice with PBS, collect the washing solution in a centrifuge tube, and store at 4°C;

[0080] (3) Prepare a culture medium containing Bafilomycin A1. Add 1 mM Bafilomycin A1 (MCE, Catalog No. HY-100558) to the preheated fresh culture medium to a final concentration of 100 nM, and incubate at 37°C, 5% CO2 for 1 h;

[0081] (4) SA-β-Gal substrate enzymatic hydrolysis: discard the old culture medium, add C12FDG to the integrated buffer to a final concentration of 33 μM, and add 10% FBS to maintain cell nutrition. Incubate at 37°C, 5% CO2 for 2 h.

[0082] (5) Digest the cells with EDTA-free trypsin and collect the cells by centrifugation at 300 g for 5 min at 4°C.

[0083] (6) Wash the cells twice with 4°C precooled PBS, centrifuge at 300g for 5 min each time, and discard the supernatant;

[0084] (7) Prepare 1× integrated buffer, resuspend the cells, and adjust the concentration to 4×106 cells / mL;

[0085] (8) Take 100 μL of the cell suspension prepared in step (7), add 5 μL of apoptotic cell fluorescent staining solution Annexin V-Alexa Fluor 647, mix well by pipetting, and incubate at room temperature in the dark for 5 min;

[0086] (9) Add 400 μL of 1× integrated buffer to each tube of sample;

[0087] (10) The samples were mixed, filtered, and detected by flow cytometry.

[0088] Test results and analysis:

[0089] The results are as follows Figure 2 and Figure 3 As shown, the kit of the present invention can be used to monitor the dynamic effects of osimertinib, a commonly used drug in the treatment of lung cancer, and doxorubicin, a chemotherapy-induced senescence drug, on the state of cancer cells under different treatment regimens. With the help of this kit, the dynamic changes in the senescence and apoptosis states of cancer cells can be accurately tracked, and double-positive cell populations, that is, those transitional cell populations that exhibit both senescence and apoptosis characteristics, can be uniquely identified. This discovery provides valuable experimental evidence and technical support for a deep understanding of the complex mechanism of cancer cell response to drug therapy, and further enriches the perspective and means of clinical research.

[0090] Example 3

[0091] The effective treatment conditions and toxicity evaluation of clinical chemotherapy drugs were explored using an integrated kit. Lung adenocarcinoma cell PC-9 was used as a model system. The safe treatment conditions of the drugs were detected and evaluated using the kit of the present invention according to the specific steps of Example 1. The specific composition of the integrated kit for detecting senescent cells and apoptotic cells used in this example is shown in Table 1. The components of the 5× integrated buffer in Table 1 include 90 mM MOPS, 770 mM NaCl, 11.5 mM CaCl2, 25 mg / ml OsrHSA, and 4.15 mM erythritol, and the pH value was adjusted to 6.0.

[0092] The specific steps are as follows:

[0093] (1) Cell culture and treatment: First, ensure that PC-9 cells are cultured to the logarithmic growth phase under appropriate conditions. Then, according to the predetermined plan, the cells are treated with a lower concentration of 50 nM doxorubicin. Cell samples of the treatment group are collected at 24 h, 48 h, and 72 h after treatment for subsequent analysis.

[0094] (2) Kit detection: According to the detailed steps in Example 1, the treated PC-9 cell samples were detected using the kit of the present invention.

[0095] (3) Other traditional detection methods: SA-β-Gal staining kit (Yisheng, catalog number 40754ES60) was used to detect the proportion of senescent cells treated with 50 nM doxorubicin for different time periods. As a non-fluorescence detection method, this kit has unique advantages in detecting the proportion of senescent cells. The results of this group were compared with the detection results of the kit of the present invention.

[0096] Test results and analysis:

[0097] Table 2 Comparison between the kit of the present invention and the Yisheng aging staining kit

[0098]

[0099] Note: For the percentage of senescent cell population in the same group of samples detected by two kits, a paired sample t-test was used to compare whether there was a statistical difference between the two groups of data, so as to evaluate the consistency and accuracy of the two kits.

[0100] The results are as follows Figure 4 and Figure 5 As shown, compared with traditional detection kits, the kit of the present invention can not only be used to evaluate the drug's senescence-inducing concentration, but also effectively evaluate the drug's potential toxicity by virtue of its built-in apoptosis detection function. Through comprehensive data analysis, it can determine the effective and safe range of conditions for doxorubicin-induced PC-9 cell senescence, which exceeds the functions of traditional detection kits.

[0101] Comparative Example 1

[0102] This comparative example aims to evaluate the specific effects of OsrHSA and erythritol in the integrated buffer on the cell detection results; four groups of lung adenocarcinoma cells of the same number were inoculated into 3.5 cm culture dishes and treated with 100 nM doxorubicin for 48 hours; then, the first group was strictly tested using the 5× integrated buffer in Table 1; the buffer used in the second group was an integrated buffer without OsrHSA; the buffer used in the third group was an integrated buffer without erythritol; the buffer used in the fourth group was an integrated buffer without OsrHSA and erythritol, so as to compare and analyze the specific effects of OsrHSA and erythritol in the buffer system. The specific steps are as follows:

[0103] (1) Collect the cell supernatant and transfer it to a centrifuge tube;

[0104] (2) Wash twice with PBS, collect the washing solution in a centrifuge tube, and store at 4°C;

[0105] (3) SA-β-Gal substrate enzymatic hydrolysis: discard the old culture medium, add C12FDG to the integrated new buffer to a final concentration of 33 μM C12FDG, and add 10% FBS to maintain cell nutrition. Incubate at 37°C in the absence of CO2 for 2 h.

[0106] (4) Digest the cells with EDTA-free trypsin, transfer the cell suspension to the centrifuge tube containing the supernatant collected in steps 2 and 3, centrifuge at 300 g, 4°C for 5 min, and discard the supernatant;

[0107] (5) Wash the cells twice with 4°C precooled PBS, centrifuge at 300g for 5 min each time, and discard the supernatant;

[0108] (6) Prepare 1× integrated buffer, resuspend the cells, and adjust the concentration to 1×10 6 cells / mL;

[0109] (7) Take 100 μL of the cell suspension prepared in step (7), add 5 μL of apoptotic cell fluorescent staining solution Annexin V-Alexa Fluor 647, mix well by pipetting, and incubate at room temperature in the dark for 5 min;

[0110] (8) Add 400 μl of 1× integrated buffer to each tube of sample;

[0111] (9) Mix the samples, filter them, and detect them by flow cytometry.

[0112] Test results and analysis:

[0113] The results are as follows Figure 6 As shown, the second group (buffer without OsrHSA) showed a decreased sensitivity in detecting senescent cell populations when compared with the first group; the third group (buffer without erythritol) showed a decreased sensitivity in detecting senescent cell populations when compared with the first group, and an increased proportion of apoptotic cell populations, which may be a false-positive cell population caused by mechanical damage during the operation; the fourth group (buffer without OsrHSA and erythritol) showed a significantly decreased sensitivity in detecting senescent cell populations, and an increased proportion of apoptotic cell populations.

[0114] Comparative Example 2

[0115] Three groups of lung adenocarcinoma cells were prepared and treated with 100 nM doxorubicin for 48 h.

[0116] The first group of cells were tested using the kit of the present invention and following the specific steps of Example 1;

[0117] The second group of cells was detected using a commercial senescence staining reagent (MCE, Cat. No. HY-126839) and processed as follows:

[0118] (1) Prepare a culture medium containing Bafilomycin A1. Add 1 mM Bafilomycin A1 (MCE, Catalog No. HY-100558) to the preheated fresh culture medium to a final concentration of 100 nM. Incubate at 37°C, 5% CO2 for 1 h.

[0119] (2) SA-β-Gal substrate enzymatic hydrolysis: discard the old culture medium, add 10% FBS, 0.1% ciprofloxacin and 1% double antibody to the DMEM culture medium, add C12FDG to a final concentration of 33 μM, and incubate at 37°C for 2 h;

[0120] (3) Wash cells twice with PBS;

[0121] (4) Digest the cells with trypsin, transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 3 min, and discard the supernatant;

[0122] (5) Resuspend the cells in PBS, filter, and detect the senescent cell population by flow cytometry.

[0123] The third group of cells was tested using a simple combination of a traditional senescence reagent (MCE, Catalog No. HY-126839) and an apoptosis kit (Tongren Chemical, Catalog No. AD10) and processed according to the following steps:

[0124] (1) Collect the cell supernatant in a centrifuge tube;

[0125] (2) Wash the cells twice with PBS and collect the washing solution into a centrifuge tube;

[0126] (3) Prepare a culture medium containing Bafilomycin A1. Add 1 mM Bafilomycin A1 (MCE, Catalog No. HY-100558) to the preheated fresh culture medium to a final concentration of 100 nM. Incubate at 37°C, 5% CO2 for 1 h.

[0127] (4) SA-β-Gal substrate enzymatic hydrolysis: discard the old culture medium, add 10% FBS, 0.1% ciprofloxacin and 1% double antibody to DMEM culture medium, add C12FDG to a final concentration of 33 μM, and incubate at 37°C for 2 h;

[0128] (5) Digest the cells with trypsin, transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 3 min, and discard the supernatant;

[0129] (6) Wash the cells twice with PBS, centrifuge at 1000 rpm for 3 min, and discard the supernatant;

[0130] (7) Add 100 μL of 1× Annexin V Binding Solution in the apoptosis kit to resuspend the cells;

[0131] (8) Add 5 μL of apoptotic cell fluorescent staining solution, Annexin V-FITC staining solution, and then add 5 μL of PI staining solution, and stain at room temperature in the dark for 15 min;

[0132] (9) Add 400 μL 1× Annexin V Binding Solution;

[0133] (10) The samples were mixed, filtered, and detected by flow cytometry.

[0134] When three different kits were used for flow cytometry detection, the negative signal range was defined, and the flow voltage and gating parameters were set according to the fluorescence signal characteristics of the blank control tube, isotype control tube and experimental samples in each kit system.

[0135] Test results and analysis:

[0136] The results are as follows Figure 7 As shown, when the samples under the same treatment conditions were tested, the following situations were observed. The results of the senescent cell population obtained by the integrated kit of the present invention and the traditional commercially available separation reagents did not show a statistically significant difference. However, compared with the test results of the simple combination of the two commercially available kits, the results of the kit of the present invention showed a statistically significant difference. This test not only confirms the high accuracy of the kit of the present invention in evaluating senescent cell populations, but also highlights the excellent sensitivity of the integrated buffer in detecting senescent cell populations.

[0137] Table 3 Comparison of the kit of the present invention with two control kits

[0138]

[0139]

[0140] Note: For the percentage of senescent cell populations in samples under the same treatment conditions detected by the two kits, a paired sample t-test was used to compare whether there was a statistical difference between the two groups of data, in order to evaluate the consistency and accuracy of the two kits.

[0141] Comparative Example 3

[0142] Three groups of lung adenocarcinoma cells were prepared and treated with 10 μM osimertinib for 24 h.

[0143] The first group of cells were tested using the kit of the present invention and following the specific steps of Example 1;

[0144] The second group of cells was tested using a commercial apoptosis kit (Tongren Chemical, Cat. No. AD10) and processed according to the following steps:

[0145] (1) Collect the cell supernatant in a centrifuge tube and store it at 4°C;

[0146] (2) Wash the cells twice with PBS and collect the washing solution into a centrifuge tube;

[0147] (3) Digest the cells with EDTA-free trypsin, transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 3 min, and discard the supernatant;

[0148] (4) Wash the cells twice with PBS, centrifuge at 1000 rpm for 3 min, and discard the supernatant;

[0149] (5) Resuspend the cells using 1× Annexin V Binding Solution in the apoptosis kit;

[0150] (6) Add 5 μL of Annexin V-FITC staining solution and 5 μL of PI staining solution, and stain for 15 min at room temperature in the dark;

[0151] (7) Add 400 μL 1× Annexin V Binding Solution;

[0152] (8) The samples were mixed, filtered, and apoptotic cell populations were detected by flow cytometry.

[0153] The third group of cells were tested using a combination of a traditional senescence reagent (MCE, Catalog No. HY-126839) and an apoptosis kit (Tongren Chemical, Catalog No. AD10) and processed according to the following steps:

[0154] (1) Collect the cell supernatant in a centrifuge tube;

[0155] (2) Wash the cells twice with PBS and collect the washing solution into a centrifuge tube;

[0156] (3) Prepare a culture medium containing Bafilomycin A1. Add 1 mM Bafilomycin A1 (MCE, Catalog No. HY-100558) to the preheated fresh culture medium to a final concentration of 100 nM. Incubate at 37°C, 5% CO2 for 1 h.

[0157] (4) SA-β-Gal substrate enzymatic hydrolysis: discard the old culture medium, add 10% FBS, 0.1% ciprofloxacin and 1% double antibody to DMEM culture medium, add C12FDG to a final concentration of 33 μM, and incubate at 37°C for 2 h;

[0158] (5) Digest the cells with trypsin, transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 3 min, and discard the supernatant;

[0159] (6) Wash the cells twice with PBS, centrifuge at 1000 rpm for 3 min, and discard the supernatant;

[0160] (7) Add 100 μL of 1× Annexin V Binding Solution in the apoptosis kit to resuspend the cells;

[0161] (8) Add 5 μL of Annexin V-FITC staining solution and 5 μL of PI staining solution, and stain for 15 min at room temperature in the dark;

[0162] (9) Add 400 μL 1× Annexin V Binding Solution;

[0163] (10) The samples were mixed, filtered, and detected by flow cytometry.

[0164] When three different kits were used for flow cytometry detection, the negative signal range was defined, and the flow voltage and gating parameters were set according to the fluorescence signal characteristics of the blank control tube, isotype control tube and experimental samples in each kit system.

[0165] Test results and analysis:

[0166] The results are as follows Figure 8 As shown, when the samples under the same treatment conditions were tested, the following situations were observed. The results of apoptotic cell populations obtained by using the integrated kit of the present invention did not show statistically significant differences compared with those obtained by using traditional commercially available separation reagents. However, compared with the test results of simply combining the two commercially available kits, the results of the kit of the present invention showed statistically significant differences. This test not only confirms the high accuracy of the kit of the present invention in evaluating apoptotic cell populations, but also highlights the excellent sensitivity of the integrated buffer in detecting apoptotic cell populations.

[0167] Table 4 Comparison of the kit of the present invention and two control kits

[0168]

[0169] Note: For the percentage of apoptotic cell populations in samples under the same treatment conditions detected by the two kits, a paired sample t-test was used to compare whether there was a statistical difference between the two groups of data, so as to evaluate the consistency and accuracy of the two kits.

[0170] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A kit for detecting senescent cells and apoptotic cells, characterized in that: It includes a senescence inducer, an apoptosis inducer, an integrated buffer, a β-galactosidase-specific fluorescent substrate, and an apoptotic cell fluorescent staining solution; The integrated buffer comprises a nonionic amphoteric buffer, sodium chloride, calcium chloride, plant-derived recombinant human serum albumin, and erythritol. The nonionic amphoteric buffer is used at a concentration of 8-18 mM, the sodium chloride is used at a concentration of 127-154 mM, the calcium chloride is used at a concentration of 2.00-2.46 mM, the plant-derived recombinant human serum albumin is used at a concentration of 0.5-5.0 mg / ml, and the erythritol is used at a concentration of 0.35-0.83 mM.

2. The integrated kit for detecting senescent cells and apoptotic cells according to claim 1, characterized in that: The nonionic amphoteric buffer is selected from one of 4-hydroxyethylpiperazineethanesulfonic acid, 2-morpholineethanesulfonic acid and 3-(N-morpholino)propanesulfonic acid.

3. The integrated kit for detecting senescent cells and apoptotic cells according to claim 1, characterized in that: The nonionic amphoteric buffer is 4-hydroxyethylpiperazineethanesulfonic acid, and the concentration of the 4-hydroxyethylpiperazineethanesulfonic acid is 10 mM.

4. The integrated kit for detecting senescent cells and apoptotic cells according to claim 1, characterized in that: The concentration of sodium chloride used is 150 mM; the concentration of calcium chloride used is 2.46 mM; the concentration of plant-derived recombinant human serum albumin used is 1 mg / ml; and the concentration of erythritol used is 0.56 mM.

5. The integrated kit for detecting senescent cells and apoptotic cells according to claim 1, characterized in that: The pH value of the integrated buffer solution is 5.8-6.4; preferably, the pH value of the integrated buffer solution is 6.

0.

6. The integrated kit for detecting senescent cells and apoptotic cells according to claim 1, characterized in that: The senescence inducing agent is doxorubicin; preferably, the senescence inducing agent is treated at a concentration of 50-200 nM for 24-96 h; preferably, the senescence inducing agent is treated at a concentration of 100 nM for 48 h.

7. The integrated kit for detecting senescent cells and apoptotic cells according to claim 1, characterized in that: The apoptosis inducer is osimertinib; preferably, the apoptosis inducing condition of the apoptosis inducer is a treatment at a concentration of 2.5-12.5 μM for 24-96 h; preferably, the apoptosis inducing condition is a treatment at a concentration of 10 μM for 48 h.

8. A method for integrated detection of senescent cells and apoptotic cells using the kit according to any one of claims 1 to 7, characterized in that: The following steps are involved: S1: Treat cell samples with senescence inducers and apoptosis inducers; S2: Collect the cell supernatant and transfer it to a centrifuge tube, wash, and collect the washing solution and store it in a centrifuge tube; Optionally, S3: add Bafilomycin A1 to pre-warmed fresh culture medium and incubate; S3: discard the old culture medium in the cell sample, add the integrated buffer supplemented with β-galactosidase-specific fluorescent substrate, and add fetal bovine serum for incubation; S4: After digesting the cells, the cell suspension is transferred to a centrifuge tube storing the supernatant collected in step S2, and the supernatant is discarded. The cells are washed, centrifuged, and the supernatant is discarded; S5: Resuspend the cells in the integrated buffer to obtain a cell suspension and adjust the cell concentration to 1-4×10 6 Pieces / mL; S6: Take the cell suspension prepared in step S5, add apoptotic cell fluorescent staining solution, mix well by pipetting, and incubate at room temperature in the dark; S7: Add the integrated buffer to the cell sample for rehydration, mix the cell sample, filter it, detect it using a flow cytometer, and collect the fluorescence signal of the cell sample.

9. Use of the kit according to any one of claims 1 to 7 in the preparation of a combined drug for treating and / or preventing tumors.

10. Use of the kit according to any one of claims 1 to 7 in evaluating the safety-inducing concentration of a drug.

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