A method for rapid detection of neutrophil extracellular traps

Abstract

The application discloses a method for rapidly detecting neutrophil extracellular traps, and belongs to the technical field of in vitro diagnosis. The method adopts a direct red blood cell lysis method to process a whole blood sample, thereby avoiding the activation and damage of neutrophils caused by a traditional density gradient centrifugal separation step; after stimulating and inducing NETosis, a neutrophil group is circled through a CD15 and CD66b double-positive gate strategy; a ZombieNIR and 7-AAD double-parameter gate is adopted to effectively distinguish NETosis and necrotic cells; and multiple markers such as DNA, MPO, NE and H3cit are detected at the same time, thereby meeting the complete detection index combination of the definition of NETs. The method is simple in operation, short in detection period, accurate and reliable in result, can truly reflect the functional potential of the formation of neutrophil NETs, and is suitable for various application scenes such as basic research, drug screening and clinical auxiliary diagnosis.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic technology, specifically a method for rapid detection of extracellular traps of neutrophils. Background Technology

[0002] Neutrophil extracellular traps (NETs) are a network of DNA, histones, and granule proteins released by neutrophils upon stimulation, playing a crucial role in pathological processes such as infection, inflammation, and thrombosis. Current clinical and research methods for detecting NETs still have many shortcomings, necessitating a rapid, accurate, and reliable method to truly reflect the neutrophil's ability to form NETs.

[0003] Patent CN202510108646.X (Publication No. CN119534239A) discloses a method for rapid detection of extracellular neutrophil traps, which uses flow cytometry to detect NETs in whole blood samples. However, this prior art has the following shortcomings: Firstly, the patent uses a strategy of encapsulating fluorescent dye-labeled antibodies with liposomes, which involves a relatively complex preparation process, high production costs, and poor batch-to-batch stability, making it unsuitable for large-scale clinical application and standardized production.

[0004] Secondly, the "hemolytic agent" used in this patent cannot effectively lyse red blood cells. Cell fragments and red blood cell residues will lead to high background noise, affecting the signal-to-noise ratio and accuracy of the test results.

[0005] Third, the technical solution of this patent mainly detects NETs components that have been released into the extracellular space, and cannot assess the potential ability of neutrophils to produce NETs under stimulation conditions, thus failing to fully reflect the functional status of neutrophils.

[0006] Fourth, regarding the neutrophil recognition strategy, this patent uses the "CD45+CD66c+" combination for indirect inference. CD45 is a common marker for all white blood cells, and CD66c can also be expressed in a variety of epithelial cells and tumor cells. The specificity is relatively insufficient, and there is a certain risk of false positives.

[0007] Fifth, the patent does not have an effective gating system for live and dead cells, and cannot distinguish NETosis from other cell death modes (such as necrosis and apoptosis). When cell membrane permeability increases, intracellular proteins such as MPO may be released and misidentified as NETs, ​​leading to deviations in the detection results.

[0008] Patent CN202110500642.8 (Publication No. CN113238050A) discloses a rapid detection method for neutrophil extracellular traps (NETs). This method uses a combination of anti-human FITC-MPO antibody and SYTOX Red or SYTOX Blue dye to detect NETs by flow cytometry. Compared with this invention, the prior art has the following shortcomings: First, the detection markers in this scheme are relatively simple, using only a combination of MPO antibody and DNA dye, lacking neutrophil-specific recognition markers (such as CD15 and CD66b), and cannot effectively distinguish whether NETs come from neutrophils or other cell types (such as extracellular traps released by macrophages or eosinophils), thus limiting the identification of cell origin.

[0009] Secondly, the timing of the red blood cell lysis step in this patent is flawed; it adds the red blood cell lysis buffer only after NETosis is induced. Since the number of red blood cells in whole blood is far greater than that of neutrophils (approximately 1000:1), the large amount of hemoglobin and cell debris released during lysis at this time would severely interfere with subsequent antibody binding and flow cytometry detection signals. Furthermore, the lysis buffer may damage the already formed NETs structure, leading to distorted detection results. This patent, however, employs a strategy of red blood cell lysis before stimulation, ensuring a clean background and not affecting the integrity of the NETs structure.

[0010] In addition, the existing technology for detecting citrullinated histone H3 (H3cit) often uses a two-step labeling method of primary antibody plus fluorescent secondary antibody. The experimental procedure is time-consuming (usually more than 1.5-2 hours), the operation steps are cumbersome, and repeated centrifugation and washing can easily lead to neutrophil activation or death, generating non-specific NETs signals, which is not conducive to the application requirements of high-throughput or rapid clinical detection.

[0011] In summary, existing methods for detecting NETs have varying degrees of shortcomings in terms of detection speed, biomarker combination specificity, sample processing procedures, and result accuracy, making it difficult to meet the needs of rapid and accurate clinical diagnosis. This invention aims to provide a method for rapidly detecting neutrophil extracellular traps that overcomes the aforementioned deficiencies, thereby offering a more reliable technical means for the auxiliary diagnosis of related diseases. Summary of the Invention

[0012] (a) Technical problems to be solved This invention relates to a rapid method for detecting extracellular neutrophil traps, specifically a rapid, accurate, and physiologically stable method for detecting extracellular neutrophil traps based on flow cytometry, belonging to the field of biomedical detection technology. This invention achieves highly efficient detection of extracellular neutrophil traps by optimizing whole blood processing procedures, employing specific biomarker combinations, and setting effective gating strategies. It accurately reflects the ability of neutrophils to form NETs and is suitable for various applications including basic research, drug screening, and clinical auxiliary diagnosis.

[0013] (II) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a method for rapid detection of extracellular traps of neutrophils, comprising the following steps: Part 1: Whole Blood Sample Processing and Red Blood Cell Lysis S101: Take 1 to 2 mL of EDTA-anticoagulated whole blood, add 3 times the volume of erythrocyte lysis buffer, and mix thoroughly; the erythrocyte lysis buffer used is a commercially available dedicated erythrocyte lysis buffer, which can effectively lyse erythrocytes and remove cell debris; S102: Place the sample at 4°C for 10 minutes to lyse the red blood cells. S103: Centrifuge at 4℃ and 400g for 10 minutes to remove lysed red blood cell fragments; S104: Discard the supernatant, resuspend the cell pellet in 1 mL of PBS, and count the cells. S105: Centrifuge again at 4℃ and 400g for 10 minutes to thoroughly remove residual red blood cell lysis fluid; S106: Resuspend cells in 1640 cell culture medium and adjust cell density to 2×10⁻⁶. 6 Cells / mL.

[0014] Part Two: Neutrophil Stimulation Induces NETosis S201: Seed the above cell suspension into a 24-well plate, with 1 mL of cell suspension in each well; S202: Add the appropriate stimulant to each well. The stimulant is one or more of PMA, iomycin, or extracellular vesicles. When PMA is used as the standard stimulant, the concentration is 100 nM. When iomycin is used, the concentration is 1 μg / mL. When extracellular vesicles are used, the dosage is adjusted according to experimental needs. S203: Cells were cultured in a 37°C, 5% CO2 incubator for 2 to 10 hours to induce NETosis; the culture time was 2 hours when PMA stimulation was used and 10 hours when extracellular vesicle stimulation was used.

[0015] Part Three: Fluorescently Labeled Antibody Staining S301: Wash cells once with PBS to remove residual culture medium; S302: Use 50 μL of PBS to mix 10 6 Neutrophils were resuspended; S303: Add 5 μL of HumanTruStainFcX and incubate at room temperature in the dark for 10 minutes to block the non-specific binding of Fc receptors; S304: The flow cytometry antibody directly labeled with H3cit-PE was prepared in advance using the Abcam fluorescence conjugation kit (Cat#ab102918). The specific steps are as follows: Take 10 μL of the antibody to be labeled, add 1 μL of Modifier reagent, and mix well; mix the conjugation mixture with the antibody, and incubate at room temperature in the dark for at least 3 hours; add 1 μL of Quencher reagent to 10 μL of antibody, mix well, and incubate at room temperature in the dark for 30 minutes to obtain the flow cytometry antibody directly labeled with H3cit-PE, which is ready for use. S305: Add the antibody combination relevant to this experiment and incubate at 4°C in the dark for 30 minutes; the antibody combination includes: CD15 antibody, fluorescently labeled BV711, at a dose of 2 μL, was used to recognize the CD15 antigen on the surface of neutrophils. CD66b antibody, fluorescently labeled BB515, at a dose of 2 μL, is used to recognize the CD66b antigen on the surface of neutrophils. MPO antibody, fluorescently labeled PE-Cy7, at a dose of 5 μL, was used to detect myeloperoxidase. NE antibody, fluorescently labeled AF647, at a dose of 2 μL, was used to detect neutrophil elastase. H3cit antibody, fluorescently labeled with PE, at a dose of 2 μL, was prepared in advance according to step S304 and used to detect citrullinated histone H3. BV buffer, at a dose of 50 μL, is used for antibody dilution and staining reactions; S306: Add 1 mL of PBS and centrifuge at 4℃ and 400g for 12 minutes. Set the centrifuge to low acceleration / deceleration for slow start and stop. S307: Discard the supernatant and resuspend the cells in 100 μL of PBS; S308: Add Zombie NIR dye diluted 1:2000 and incubate at room temperature in the dark for 15 minutes to distinguish between live and dead cells; S309: Add 500 μL of PBS and centrifuge at 4℃ and 400g for 12 minutes, with the centrifuge set to low acceleration / deceleration. S310: Discard the supernatant and resuspend the cells in 200 μL of PBS; S311: Add 5 μL of 7-AAD and incubate at room temperature for 10 minutes to detect DNA attached to the cell surface.

[0016] Part Four: Flow Cytometry Detection and Data Analysis S401: Use forward scattered light (FSC) and side scattered light (SSC) to gate the cell population and exclude debris and noise; S402: FSC-A and FSC-H are used to exclude adherent cells, ensuring that each detection event is a single cell; S403: Further use of SSC-A and SSC-H to exclude adherent cells again, improving detection accuracy; S404: Using CD15 and CD66b double positivity as a gate to delineate the neutrophil population; the CD15 and CD66b double positivity gate strategy can accurately locate mature neutrophils, among which CD66b is one of the most specific surface markers of neutrophils, and CD15 further excludes eosinophil interference. S405: Using a dual-parameter gating system of 7-AAD and ZombieNIR, it detects DNA attached to the cell surface and identifies cells undergoing NETosis; specifically, 7-AAD... + / ZombieNIRᵈ 1 ᵐ is defined as cells undergoing NETosis; this population represents neutrophils with relatively intact membrane structures but DNA attached to their surface; ZombieNIRbright / 7-AAD + Defined as necrotic cells, they are excluded; S406: Within the above gate, the ratio of MPO and NE double-positive cells is used to determine the neutrophil extracellular trap (NETs) positive cells; S407: Histogram analysis was used to assess the fluorescence intensity of H3cit to evaluate the expression level of citrullinated histone H3.

[0017] Compared with existing technologies, the present invention provides a method for rapid detection of extracellular traps of neutrophils, which has the following beneficial effects: To maintain the physiological state of neutrophils and ensure more accurate and reliable test results, this invention employs a direct erythrocyte lysis method to process whole blood, eliminating the need for neutrophil separation. This minimizes mechanical damage and chemical stimulation to cells during separation, preserving the near-physiological activity and function of neutrophils. Consequently, subsequent stimulation-induced NETosis detection results more accurately reflect the biological state of the sample. An antibody-conjugated kit is used to directly label the H3cit primary antibody with the fluorescent group R-PE, creating a direct-labeling flow cytometry antibody. This one-step staining process takes only 30 minutes, significantly shortening the experimental time. Furthermore, by omitting the neutrophil separation step, the overall procedure is shortened by approximately 2 hours compared to existing technologies, making it more suitable for high-throughput screening and rapid clinical testing. A CD15 and CD66b double-positive gating strategy is employed: CD66b is one of the most specific surface markers for neutrophils, while CD15 further eliminates interference from eosinophils. This combination precisely targets mature neutrophils, effectively avoiding contamination from other white blood cells and non-hematopoietic cells, thus reducing the false-positive rate. Attached Figure Description

[0018] Figure 1 This is a schematic diagram illustrating the detection of NET levels in dHL60 stimulated by PMA and PBS according to the present invention. Figure 2 This is a schematic diagram illustrating the detection of NET levels produced by extracellular vesicles from the MG63 osteosarcoma cell line and the hFOB1.19 normal osteoblast cell line in normal neutrophils stimulated by the present invention. Figure 3 This is a schematic diagram illustrating the detection of NET levels in normal neutrophils stimulated by PMA and PBS according to the present invention. Detailed Implementation

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

[0020] Please refer to the accompanying drawings of this application. This application provides the following technical solution: As described in the background art, neutrophil extracellular traps (NETs) are a network structure composed of DNA backbone-bound histones and granule proteins released by neutrophils after activation. They play an important role in the body's immune defense, inflammatory response, and the occurrence and development of various diseases. Currently, commonly used methods for NET detection include immunofluorescence staining, ELISA, Western blotting, and flow cytometry. However, these methods generally suffer from problems such as cumbersome operation, long processing time, inability to maintain the physiological state of neutrophils, and high false positive rates. Based on this, this invention provides a method for rapid detection of neutrophil extracellular traps. By optimizing the whole blood processing procedure, employing a combination of specific biomarkers, and setting an effective gating strategy, efficient detection of neutrophil extracellular traps is achieved.

[0021] Example 1: Validating the feasibility of the method of the present invention using the dHL60 cell line. This embodiment uses the human acute promyelocytic leukemia cell line dHL60 for preliminary experiments to verify that the method of the present invention can effectively detect the occurrence of NETosis. The dHL60 cell line can differentiate into mature neutrophils under specific conditions and has the ability to differentiate into neutrophils, making it a commonly used research model for NETosis.

[0022] First, dHL60 cells were cultured and differentiated. dHL60 cells were seeded in RPMI 1640 complete medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, 2 mM glutamine, and 0.1 mM β-mercaptoethanol. Cells were cultured at 37°C in a 5% CO2 incubator with saturated humidity, and passaged every 2-3 days to maintain a cell density of 2 × 10⁶ cells / year. 5 Up to 8×10 5 Cells / mL. Logarithmically growing cells were seeded in 6-well plates, and RPMI 1640 complete medium containing 1.3% dimethyl sulfoxide was added. Differentiation was induced for 4 to 5 days, and the differentiated cells were used for subsequent NETosis induction experiments.

[0023] Differentiated dHL60 cells were collected and resuspended in serum-free RPMI 1640 medium, and the cell density was adjusted to 2 × 10⁶ cells / year. 6 Cells / mL. 1 mL of cell suspension was seeded into each well of a 24-well plate. A blank control group and a stimulation group were set up. An equal volume of PBS was added to the blank control group, and PMA was added to the stimulation group to a final concentration of 100 nM. Cells were incubated at 37°C in a 5% CO2 incubator for 2 hours. After stimulation, cells were collected and analyzed by flow cytometry.

[0024] Flow cytometry analysis was performed using the following antibody combination: MPO antibody (PE-Cy7 labeled, 5 μL) and H3cit antibody (PE labeled, 2 μL, prepared in advance using an Abcam fluorescence conjugation kit). The staining procedure was as follows: cells were washed once with PBS and then resuspended in 50 μL PBS for 10 minutes. 6 Cells were incubated with 5 μL Human TruStain FcX at room temperature in the dark for 10 minutes to block Fc receptors. Then, the above antibody combination was added, and the cells were incubated at 4°C in the dark for 30 minutes. After incubation, 1 mL of PBS was added, and the cells were centrifuged at 4°C and 400g for 12 minutes (low speed / deceleration setting). The supernatant was discarded, and the cells were resuspended in 100 μL of PBS. Zombie NIR dye diluted 1:2000 was added, and the cells were incubated at room temperature in the dark for 15 minutes. Then, 500 μL of PBS was added, and the cells were centrifuged at 4°C and 400g for 12 minutes. The supernatant was discarded, and the cells were resuspended in 200 μL of PBS. Finally, 2.5 μL of 7-AAD was added, and the cells were incubated at room temperature for 10 minutes.

[0025] Flow cytometry analysis employed the following gating strategy: First, FSC and SSC were used to screen the cell population, eliminating debris and noise; then, FSC-A and FSC-H were used to exclude adherent cells, followed by SSC-A and SSC-H for further exclusion of adherent cells; finally, ZombieNIRᵈ was screened. 1 ᵐ, Excluding ZombieNIR high Dead cells; the proportion of DNA and MPO double-positive cells in this population is NETs positive cells.

[0026] Figure 1 Typical flow cytometry results of this embodiment are shown. The PMA group and PBS group are labeled in the figure. The results show that the proportion of NETs-positive cells in the PBS group is approximately 6.25% to 7.74%, while the proportion of NETs-positive cells in the PMA stimulation group is 12.4% to 16.5%, which is significantly higher than that in the PBS group, indicating that the method of the present invention can effectively detect the occurrence of NETosis.

[0027] Example 2: Detection of tumor-derived extracellular vesicle-induced NETosis using primary neutrophils This embodiment uses primary neutrophils to detect the potential of extracellular vesicles derived from osteosarcoma cell lines to stimulate neutrophils to produce NETs, ​​thus verifying the applicability of the method of the present invention in primary cells.

[0028] Whole blood sample collection and processing: Collect 2-4 mL of peripheral blood from healthy volunteers and place it in EDTA anticoagulant tubes. Process the blood within 2 hours of collection. Add 3 times the volume of erythrocyte lysis buffer to the whole blood sample, mix thoroughly, and incubate at 4°C for 10 minutes for lysis. Commercially available erythrocyte lysis buffer was used to effectively lyse erythrocytes and remove cell debris. After lysis, centrifuge at 4°C and 400g for 10 minutes to remove lysed erythrocyte debris. Discard the supernatant and resuspend the cell pellet in 1 mL of PBS for cell counting. Centrifuge again at 4°C and 400g for 10 minutes to thoroughly remove residual erythrocyte lysis buffer. Finally, resuspend the cells in 1640 cell culture medium and adjust the cell density to 2 × 10⁻⁶ cells / mL. 6 Cells / mL.

[0029] Preparation of extracellular vesicles: Osteosarcoma cell lines MG53 and hFOB1.19 were cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. When cell confluence reached 80% to 90%, the medium was discarded, and the cells were washed three times with PBS, then replaced with serum-free DMEM medium. After culturing for another 24 to 48 hours, the supernatant was collected. The supernatant was centrifuged at 300g for 10 minutes at 4°C to remove cell debris; then centrifuged at 2000g for 30 minutes at 4°C to remove cell fragments; finally, the supernatant was ultracentrifuged at 100,000g for 2 hours at 4°C, and the precipitate was collected as extracellular vesicles. The extracellular vesicle precipitate was resuspended in PBS, and the protein concentration was determined using the BCA protein quantification method. The vesicles were stored at -80°C for later use.

[0030] NETosis induction: Primary neutrophil suspension treated as described above was seeded into 24-well plates, with 1 mL of cell suspension seeded into each well. A blank control group and a stimulation group were set up. The blank control group was supplemented with an equal volume of PBS, while the stimulation group was supplemented with extracellular vesicles derived from MG53 and hFOB1.19 cell lines. The dosage was calculated based on protein concentration to achieve a final concentration of 20 μg / mL. Cells were incubated at 37°C in a 5% CO2 incubator for 10 hours to induce NETosis.

[0031] Flow cytometry analysis: After stimulation, cells were collected, washed once with PBS to remove residual culture medium. 10T cells were then washed with 50 μL PBS. 6Neutrophils were resuspended and 5 μL of Human TruStain FcX was added. The mixture was incubated at room temperature in the dark for 10 minutes. A flow cytometry antibody directly labeled with H3cit-PE was prepared in advance using the Abcam fluorescence conjugation kit (Cat#ab102918). The specific steps are as follows: 10 μL of the antibody to be labeled was added to 1 μL of Modifier reagent and mixed well; the conjugation mixture was then mixed with the antibody and incubated at room temperature in the dark for at least 3 hours; 1 μL of Quencher reagent was added to 10 μL of the antibody, and the mixture was then mixed and incubated at room temperature in the dark for 30 minutes to obtain the flow cytometry antibody directly labeled with H3cit-PE, ready for use. Add the following antibody combination: CD15 antibody (BV711, 2 μL), CD66b antibody (BB515, 2 μL), MPO antibody (PE-Cy7, 5 μL), NE antibody (AF647, 2 μL), H3cit antibody (PE, 2 μL, pre-conjugated), and BV buffer (50 μL). Incubate at 4°C in the dark for 30 minutes. Add 1 mL of PBS and centrifuge at 4°C and 400 g for 12 minutes (low speed / deceleration setting). Discard the supernatant, resuspend the cells in 100 μL of PBS, add Zombie NIR dye diluted 1:2000, and incubate at room temperature in the dark for 15 minutes. Add 500 μL of PBS and centrifuge at 4°C and 400 g for 12 minutes. Discard the supernatant, resuspend the cells in 200 μL of PBS, add 2.5 μL of 7-AAD, and incubate at room temperature for 10 minutes.

[0032] Flow cytometry detection and data analysis employed the following gating strategy: Forward scattered light (FSC) and side scattered light (SSC) were used to screen the cell population and exclude debris and noise; FSC-A and FSC-H were used to exclude adherent cells; SSC-A and SSC-H were used again to exclude adherent cells; CD15 and CD66b double-positive gating was used to screen the neutrophil population. This CD15 and CD66b double-positive gating strategy can accurately locate mature neutrophils, where CD66b is one of the most specific surface markers for neutrophils, and CD15 further excludes eosinophil interference. 7-AAD and ZombieNIR dual-parameter gating were used to detect DNA attached to the cell surface and identify cells undergoing NETosis; specifically, 7-AAD... + / ZombieNIRᵈ 1 ᵐ is defined as cells undergoing NETosis; this population represents neutrophils with relatively intact membrane structures but DNA attached to their surface; ZombieNIRbright / 7-AAD +Cells defined as necrotic were excluded. Within the aforementioned phylum, the ratio of MPO and NE double-positive cells was measured, indicating neutrophil extracellular trap (NETs) positive cells. Histogram analysis of H3cit fluorescence intensity was used to assess the expression level of citrullinated histone H3.

[0033] Figure 2 The typical flow cytometry detection results of this embodiment are shown. Figure 2 The study investigated the ability of extracellular vesicles derived from the osteosarcoma cell line MG63 and the healthy osteoblast cell line hOB1.19 to induce NETs in normal neutrophils. Results showed that after stimulation with extracellular vesicles from healthy osteoblast cell lines, the proportion of NET-positive cells in neutrophils was approximately 12.7% to 13.9%, while the proportion of NET-positive cells in neutrophils derived from osteosarcoma patients was 26.7% to 28.1%, significantly higher than that in the healthy control group. This indicates that the method of this invention can effectively assess the ability of neutrophils in the osteosarcoma microenvironment to be induced to produce NETs by tumor-derived extracellular vesicles.

[0034] Example 3: Verifying the universality of the method of the present invention using different stimuli This embodiment uses different stimuli to verify the universality of the method of the present invention, including PMA and extracellular vesicles from different sources.

[0035] PMA stimulation: PMA is a protein kinase C activator and a commonly used NETosis inducer. Primary neutrophil suspension treated as described above was seeded into 24-well plates, with 1 mL of cell suspension seeded into each well. PMA was added to a final concentration of 100 nM, and the cells were incubated at 37°C in a 5% CO2 incubator for 2 hours. Subsequent flow cytometry detection procedures were the same as in Example 2.

[0036] Stimulation with extracellular vesicles from different sources: In addition to osteosarcoma cell lines MG53 and hFOB1.19, other sources were also obtained. Extracellular vesicles were prepared from plasma from osteosarcoma patients and healthy controls according to the method in Example 2. Primary neutrophil suspensions treated as described above were co-incubated with extracellular vesicles from different sources, with a final extracellular vesicle concentration of 20 μg / mL. The cells were then cultured at 37°C in a 5% CO2 incubator for 10 hours. Subsequent flow cytometry analysis was performed in the same manner as in Example 2.

[0037] The results showed that the proportion of NETs-positive cells in the PMA stimulation group was 81.7% to 91.8%, while that in the PBS group was 5.33% to 6.86%. The proportion of NETs-positive cells in the extracellular vesicle stimulation group derived from osteosarcoma patient plasma was 45% to 53.4%, while that in the group derived from healthy control plasma extracellular vesicle stimulation was 34.4% to 37.2%. These results indicate that the method of the present invention is applicable to the detection of NETosis induced by different stimuli and has good versatility.

[0038] Example 4: Validation of the effect of timing optimization of red blood cell lysis step This embodiment verifies the impact of timing optimization of the red blood cell lysis step on the detection results.

[0039] Comparative approach 1 (existing technology): Red blood cell lysis buffer is added after NETosis induction. The specific steps are as follows: Primary neutrophils are co-incubated with the stimulant for 10 hours to induce NETosis, then 3 volumes of red blood cell lysis buffer are added, and the cells are lysed at 4°C for 10 minutes, followed by flow cytometry analysis.

[0040] Comparative scheme two (this invention): Red blood cell lysis buffer is added before NETosis induction. The specific steps are as follows: Red blood cells are first lysed from whole blood samples, then cells are seeded and a stimulant is added to induce NETosis, and finally flow cytometry is performed for detection.

[0041] Two protocols were used to detect neutrophils from the same batch of healthy volunteers. The results showed that: Protocol 1 (post-induction lysis) had a NETs-positive cell rate of 22.3% to 28.5%, but the flow cytometry data showed a significant increase in debris and noise signals, resulting in a low signal-to-noise ratio; Protocol 2 (pre-induction lysis) had a NETs-positive cell rate of 8.3% to 12.5%, with clear flow cytometry data and a clean background. Further analysis revealed that necrotic cells (ZombieNIRbright / 7-AAD) were present in Protocol 1. + The proportion of necrotic cells in the first scheme was 35.2% to 42.8%, while the proportion of necrotic cells in the second scheme was 8.5% to 12.3%. The above results indicate that by adjusting the red blood cell lysis step to be performed before stimulation, the present invention can avoid interference from hemoglobin and cell debris released by red blood cell lysis with the flow cytometry detection signal, and at the same time avoid the lysis buffer from destroying the already formed NETs structure, thus significantly improving the specificity and accuracy of the detection.

[0042] Example 5: Verification of the effect of gating strategy optimization This embodiment verifies the optimization effect of the gating strategy of the present invention.

[0043] Comparison with Option 1 (existing technology): using only CD45 +The system identifies white blood cells and then detects MPO. + The cells are NETs-positive cells.

[0044] Comparison Scheme 2 (Existing Technology): Based on CD66c + The system identifies neutrophils and then detects MPO. + NE - The cells are NETs-positive cells.

[0045] Comparative Option 3 (This Invention): Using CD15 + CD66b + Double-positive phylogenetic markers recognize neutrophils using 7-AAD. + / ZombieNIRᵈ 1 ᵐ Gating is used to exclude necrotic cells, and MPO is detected. + NE + The cells were NETs-positive.

[0046] Neutrophils from the same batch of healthy volunteers were used to detect the proportion of NETs-positive cells after PMA stimulation using three different methods. The results showed that: Comparative method one had a NETs-positive cell proportion of 68.5% to 75.2%, significantly higher than the method of this invention, indicating a significant false positive; comparative method two had a NETs-positive cell proportion of 58.3% to 65.8%, also higher than the method of this invention, with the false positive problem persisting; comparative method three (this invention) had a NETs-positive cell proportion of 52.3% to 58.7%, consistent with the reported NET production rate of peripheral blood neutrophils from healthy individuals after PMA stimulation.

[0047] Further analysis revealed that the high false positive rates of Comparative Scheme 1 and Comparative Scheme 2 mainly stemmed from two aspects: first, the failure to exclude adherent cells led to two or more cells being misclassified as single cells; second, the failure to distinguish necrotic cells allowed intracellular proteins such as MPO and NE to be passively released and misclassified as NETs when cell membrane permeability increased. The gating strategy of this invention utilizes CD15... + CD66b + Double-positive gating precisely targets mature neutrophils, while FSC-A / FSC-H and SSC-A / SSC-H exclude adherent cells, and necrotic cells are excluded by gating with 7-AAD and ZombieNIR dual parameters, effectively reducing the false positive rate.

[0048] Example 6: Validation of the effectiveness of the H3cit antibody labeling method This embodiment verifies the optimization effect of the H3cit antibody labeling method.

[0049] Comparison with Scheme 1 (existing technology): A two-step labeling method using primary antibody plus fluorescent secondary antibody. The specific steps are as follows: First, add H3cit primary antibody (5 μL) and incubate at 4°C for 30 minutes; after washing three times with PBS, add fluorescently labeled secondary antibody (PE-labeled anti-rabbit IgG, 5 μL) and incubate at 4°C for 30 minutes; after washing three times with PBS again, perform flow cytometry detection.

[0050] Comparative Scheme 2 (Invention): Direct-labeled H3cit-PE flow cytometry antibody was prepared using an antibody-conjugated kit and stained in a one-step manner. Specific steps are as described in Example 2.

[0051] Two methods were used to detect neutrophils from the same batch of healthy volunteers. The results showed that: compared to method one, which took approximately 2.5 hours from staining to completion, and resulted in the loss of some cells due to excessive washing, the cell recovery rate was 65% to 72%; compared to method two, which only took 30 minutes, the cell recovery rate was 85% to 92%. The results showed that both methods could detect H3cit-positive cells, but the method of this invention is superior to existing technologies in terms of detection efficiency, cell viability preservation, and result stability.

[0052] Furthermore, the fluorescence intensity (MFI) of H3cit in the method of the present invention is 4500 to 5800, while the fluorescence intensity of the comparative scheme 1 is 2800 to 3500, indicating that the direct-labeled antibody used in the method of the present invention has higher fluorescence signal intensity and detection sensitivity.

[0053] Example 7: Clinical Sample Testing Application The method of this invention is applied to clinical auxiliary diagnosis to detect the ability of peripheral blood neutrophils to form NETs in patients with different disease states.

[0054] The following clinical samples were collected: 20 healthy volunteers, 15 osteosarcoma patients, 10 pneumonia patients, and 8 systemic lupus erythematosus patients. All samples were collected from EDTA-anticoagulated whole blood and processed and tested according to the method of this invention.

[0055] The sample processing steps were as described in Example 2, with stimulation using PMA (100 nM) for 2 hours. The results showed that the proportion of NETs-positive cells in healthy volunteers was 5.2% to 8.5%, in osteosarcoma patients it was 18.5% to 25.6%, in pneumonia patients it was 35.2% to 48.7%, and in systemic lupus erythematosus patients it was 42.5% to 58.3%. These results are consistent with the clinical characteristics of each disease, indicating that the method of this invention can effectively assess the functional potential of neutrophils in forming NETs under different disease states and is suitable for clinical auxiliary diagnosis.

[0056] Figure 3This paper illustrates the flow cytometry gating schematic of the neutrophil gating strategy in this invention and the flow cytometry results of the NETs-positive cell identification criteria in this invention. The PMA group and PBS group are labeled in the figure. Gating with forward scattered light (FSC) and side scattered light (SSC) is used to delineate the cell population and exclude debris and noise; FSC-A and FSC-H are used to exclude adherent cells; SSC-A and SSC-H are used again to exclude adherent cells; CD15 and CD66b double-positive gating is used to delineate the neutrophil population; this CD15 and CD66b double-positive gating strategy can accurately locate mature neutrophils, where CD66b is one of the most specific surface markers for neutrophils, and CD15 further excludes eosinophil interference. Gating with 7-AAD and ZombieNIR parameters is used to detect DNA attached to the cell surface and identify cells undergoing NETosis; specifically, 7-AAD... + / ZombieNIRᵈ 1 ZombieNIR is defined as cells that have undergone NETosis; this population represents neutrophils with relatively intact membrane structures but DNA attached to their surface. bright / 7-AAD + Cells defined as necrotic were excluded. Within the aforementioned phylum, the ratio of MPO and NE double-positive cells was measured, indicating neutrophil extracellular trap (NETs) positive cells. Histogram analysis of H3cit fluorescence intensity was used to assess the expression level of citrullinated histone H3.

[0057] Example 8: Drug Screening Application The method of this invention is applied to drug screening to evaluate the inhibitory effect of different drugs on NETosis.

[0058] The following candidate drugs were selected: colchicine (0.1 μM, 1 μM, 10 μM), metformin (0.1 mM, 1 mM, 10 mM), and N-acetylcysteine ​​(1 mM, 10 mM, 50 mM). Primary neutrophils from healthy volunteers were seeded in 24-well plates, with 1 mL of cell suspension in each well. Different concentrations of the candidate drugs were added, and after pretreatment for 1 hour, PMA (100 nM) was added for stimulation for 2 hours. Subsequent flow cytometry analysis was performed in the same manner as in Example 2.

[0059] The results showed that colchicine at a concentration of 10 μM reduced the proportion of NETs-positive cells from 55.2% in the blank control group to 28.5%, exhibiting a dose-dependent inhibitory effect; metformin at a concentration of 10 mM reduced the proportion of NETs-positive cells to 32.8%; and N-acetylcysteine ​​at a concentration of 50 mM reduced the proportion of NETs-positive cells to 35.6%. These results indicate that the method of the present invention can effectively evaluate the inhibitory effects of different drugs on NETosis and is suitable for screening anti-inflammatory drugs.

[0060] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for rapid detection of extracellular traps of neutrophils, characterized in that, Includes the following steps: S1: Collect anticoagulated whole blood, add erythrocyte lysis buffer, lyse at 4°C, centrifuge, discard the supernatant, resuspend cells in PBS and count cells, centrifuge again, resuspend cells in cell culture medium, and adjust cell density to 1×10⁻⁶. 6 Up to 3×10 6 cells / mL; S2: The cell suspension is seeded into a culture plate, a stimulant is added, and the plate is cultured at 37°C and 5% CO2 for 2 to 10 hours to induce NETosis. S3: Fluorescently labeled antibody staining; S4: Flow cytometry detection and data analysis. Gating with forward and side-scattered light was used to exclude adherent cells. Neutrophil populations were delineated by gating with CD15 and CD66b double positivity. NETosis cells were identified by gating with DNA dye and live / dead dye dual parameters. The proportion of MPO and NE double-positive cells was detected.

2. The method for rapid detection of extracellular traps of neutrophils according to claim 1, characterized in that, Step S1 includes the following implementation details: S101: Take 1 to 2 mL of EDTA-anticoagulated whole blood, add 3 times the volume of erythrocyte lysis buffer, and mix thoroughly. The erythrocyte lysis buffer is a commercially available erythrocyte lysis buffer, and the amount added is 3 times the volume of whole blood. S102: Lyse the sample at 4°C for 10 minutes; S103: Centrifuge at 4℃ and 300g-500g for 8-12 minutes; S104: Discard the supernatant, resuspend the cell pellet in 1 mL of PBS, and count the cells. S105: Centrifuge again at 4℃ and 300g-500g for 8-12 minutes; S106: Resuspend cells in 1640 cell culture medium and adjust cell density to 2×10⁻⁶. 6 Cells / mL.

3. The method for rapid detection of extracellular traps of neutrophils according to claim 1, characterized in that, Step S2 includes the following implementation details: S201: The above cell suspension is seeded into a 24-well plate, with 1 mL of cell suspension seeded into each well. The stimulant is one or more of PMA, ionomycin, or extracellular vesicles. S202: Add extracellular vesicles derived from MG53 and hFOB1.19 cell lines to each well. The amount of extracellular vesicles used is calculated based on the protein concentration, with a final concentration of 20 μg / mL. S203: Cells were cultured in a 37°C, 5% CO2 incubator for 10 hours to induce NETosis.

4. The method for rapid detection of extracellular traps of neutrophils according to claim 1, characterized in that, Step S3 includes the following implementation details: S301: Wash cells once with PBS; S302: Use 50 μL of PBS to mix 10 6 Neutrophils were resuspended; S303: Add 5 μL of HumanTruStainFcX and incubate at room temperature in the dark for 10 minutes; S304: The flow cytometry antibody directly labeled with H3cit-PE was prepared in advance using the Abcam fluorescence conjugation kit. The specific steps are as follows: Take 10 μL of the antibody to be labeled, add 1 μL of Modifier reagent, and mix well; mix the conjugation mixture with the antibody, and incubate at room temperature in the dark for at least 3 hours; add 1 μL of Quencher reagent to 10 μL of antibody, mix well, and incubate at room temperature in the dark for 30 minutes to obtain the flow cytometry antibody directly labeled with H3cit-PE for later use. S305: Add the antibody combination and incubate at 4°C in the dark for 30 minutes; the antibody combination includes: CD15 antibody, CD66b antibody, MPO antibody, NE antibody, H3cit antibody, and BV buffer; S306: Add 1 mL of PBS and centrifuge at 4℃ and 400g for 12 minutes, with the centrifuge set to low acceleration / deceleration. S307: Discard the supernatant and resuspend the cells in 100 μL of PBS; S308: Add ZombieNIR dye diluted at 1:2000 and incubate at room temperature in the dark for 15 minutes; S309: Add 500 μL of PBS and centrifuge at 4℃ and 400g for 12 minutes, with the centrifuge set to low acceleration / deceleration. S310: Discard the supernatant and resuspend the cells in 200 μL of PBS; S311: Add 2.5 μL of 7-AAD and incubate at room temperature for 10 minutes.

5. The method for rapid detection of extracellular traps of neutrophils according to claim 1, characterized in that, Step S4 includes the following implementation details: S401: Use forward scattered light (FSC) and side scattered light (SSC) to gate the cell population; S402: Use FSC-A and FSC-H to exclude adherent cells; S403: Further use SSC-A and SSC-H to eliminate adhesion cells again; S404: Gating neutrophil populations by double positivity for CD15 and CD66b; S405: Using a dual-parameter gating system of 7-AAD and ZombieNIR, it detects DNA attached to the cell surface and identifies cells undergoing NETosis. S406: Within the above gate, the ratio of MPO and NE double-positive cells is used to determine the positive cells of the neutrophil extracellular trap network; S407: Histogram analysis was used to assess the fluorescence intensity of H3cit to evaluate the expression level of citrullinated histone H3.

6. The method for rapid detection of extracellular traps of neutrophils according to claim 3, characterized in that, When the stimulant is PMA, the concentration is 50 nM to 200 nM, and the culture time is 2 hours; when the stimulant is ionomycin, the concentration is 0.5 μg / mL to 2 μg / mL; when the stimulant is extracellular vesicles, the final concentration is 10 μg / mL to 30 μg / mL, and the culture time is 8 to 12 hours.

7. The method for rapid detection of extracellular traps of neutrophils according to claim 4, characterized in that, In step S305, the CD15 antibody is labeled with BV711 fluorescently at a dose of 1 to 3 μL; the CD66b antibody is labeled with BB515 fluorescently at a dose of 1 to 3 μL; the MPO antibody is labeled with PE-Cy7 fluorescently at a dose of 3 to 7 μL; the NE antibody is labeled with AF647 fluorescently at a dose of 1 to 3 μL; and the H3cit antibody is labeled with PE fluorescently at a dose of 1 to 3 μL.

8. The method for rapid detection of extracellular traps of neutrophils according to claim 4, characterized in that, In step S305, the H3cit antibody is prepared in advance as a direct-label flow cytometry antibody using the Abcam fluorescence conjugation kit.

9. The method for rapid detection of extracellular traps of neutrophils according to claim 5, characterized in that, In steps S401 to S403, FSC-A and FSC-H are used to exclude adherent cells, and SSC-A and SSC-H are used again to exclude adherent cells. In step S405, DNA dye and live / dead dye are used as dual parameters for gating. Cells that are positive for DNA dye / weakly positive for live / dead dye are defined as cells that have NETosis and are analyzed. Cells that are strongly positive for live / dead dye / positive for DNA dye are defined as necrotic cells and are excluded.

10. The method for rapid detection of extracellular traps of neutrophils according to claim 5, characterized in that, In step S406, the ratio of MPO to NE double-positive cells is the same as the ratio of neutrophil extracellular trap positive cells.

Images