Method for inducing neutrophil to generate NETs
Through the combined use of cytokines IL-1β, IFN-γ and GM-CSF, the problems of carcinogenicity and high concentration induced by neutrophils in the prior art were solved, and low toxicity and efficient NET induction were achieved, and simulation The microenvironment in vivo has improved the accuracy and safety of induction.
Patent Information
- Application Number
- CN202411433426.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-14
- Publication Date
- 2024-12-31
AI Technical Summary
The existing methods of inducing NETs to neutrophils have problems such as carcinogenicity, cytotoxicity or high concentrations, and exogenous inducible substances cannot simulate the microenvironment in vivo, limiting their effectiveness and safety induction in vitro.
The isolation and purification of human peripheral blood neutrophils and the combination of cytokines IL-1β, IFN-γ and GM-CSF were used to induce neutrophil production NETs, and immunofluorescence and enzyme-linked immunosorbent assay were used to determine whether the cytokines IL-1β, IFN-γ and GM-CSF were used to induce neutrophil production. Detect induction effects.
It has achieved efficient induction of neutrophil production of NETs at low toxicity and low concentrations, which has improved the accuracy and safety of induction, and can better simulate the microenvironment in vivo.
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Abstract
Description
Technical Field
[0001] The present invention involves multiple fields such as immunology, cell biology, molecular biology, etc. Specifically, it includes separation and purification of neutrophils, and inducing neutrophils to produce neutrophil extracellular traps (NETs) by combining cytokines IL-1β, IFN-γ and GM-CSF. Background Art
[0002] Neutrophils are the most abundant innate immune effector cells in the human immune system. They are usually the first white blood cells recruited to the site of inflammation and can eliminate pathogens and promote damage repair through a variety of mechanisms. Neutrophils are traditionally considered to be effector cells of inflammatory responses and acute immunity. They function through intracellular phagocytosis and use lytic proteases, reactive oxygen radicals (ROS), etc. to kill pathogenic microorganisms. Recent studies have also shown that in addition to phagocytosis, neutrophils can adhere to and eliminate pathogenic microorganisms by releasing NETs, and can effectively limit the spread of pathogens to other tissue sites. NETs are an extracellular structure that is a mesh structure composed of decondensed chromatin and neutrophil-derived nuclear proteins, cytoplasmic proteins, and granular proteins. These proteins are embedded in the DNA backbone and greatly increase their local concentration.
[0003] Currently, there are many types of inducers of NETs. In addition to microorganisms, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), glucose oxidase (GO), tumor necrosis factor α (TNF-α), etc. can stimulate neutrophils to produce NETs to a certain extent.
[0004] However, the existing methods for inducing NETs in vitro either have strong carcinogenicity and cytotoxicity (such as PMA), or require high concentrations to have good induction ability (such as LPS), or the chemical structure of the drug itself is unstable (such as GO). At the same time, PMA is an exogenous inducing substance that is not secreted by the cells themselves and cannot better simulate the in vivo microenvironment.
[0005] Therefore, although PMA and other inducers are often used as inducers due to their obvious effects in inducing neutrophils to produce NETs in vitro, there are still many limitations. Therefore, seeking a stable and effective method to induce neutrophils to produce NETs, as well as low-toxicity, low-concentration inducers, will help to induce NETs more accurately and efficiently. Summary of the invention
[0006] The present invention provides a method for inducing neutrophils to produce NETs, the steps of which include:
[0007] 1. Isolation and purification of neutrophils from human peripheral blood:
[0008] 1) Take fresh anticoagulated whole blood and dilute it with sterile PBS of equal ratio.
[0009] 2) Add an appropriate amount of Ficoll separation solution to the centrifuge tube (when the blood volume after dilution is less than 3mL, add 3mL of separation solution; if it is greater than or equal to 3mL, add an equal volume of separation solution. However, the total volume of the two cannot exceed two-thirds of the centrifuge tube, otherwise the separation effect will be affected), spread the diluted blood evenly above the surface of the separation solution, and pay attention to keeping the interface between the two liquid surfaces clear.
[0010] 3) Centrifuge at 2000 rpm for 20 min at room temperature.
[0011] 4) After centrifugation, obvious stratification will appear: the top layer is the diluted plasma layer, the middle layer is the transparent separation liquid layer, the white film layer between the plasma and the separation liquid is the lymphocyte layer, and the bottom of the centrifuge tube is the red blood cells and granulocytes.
[0012] 5) Discard the liquid above the red blood cell and granulocyte precipitates, wash the precipitates with 10 mL of PBS, and centrifuge at 2000 rpm for 5 min.
[0013] 6) Discard the supernatant, add 6 mL of red blood cell lysis buffer, lyse for 5 minutes, and then centrifuge at 1800 rpm for 5 minutes.
[0014] 7) Repeat step 6) once.
[0015] 8) Add 10 mL of PBS, wash and resuspend the cells to obtain neutrophils.
[0016] 9) Discard the supernatant and add a certain amount of RPMI 1640 complete medium to resuspend the cells for later use.
[0017] 10) Flow cytometry was used to detect the purity of neutrophils (using CD45, CD66b and CD16 as indicators).
[0018] 2. Combined use of cytokines to treat neutrophils:
[0019] 1) Cells were extracted as described in 1. According to the experimental design, 10 ng / mL IFN-γ, 1 ng / mL GM-CSF and 30 ng / mL IL-1β were added to 200 μL / well of neutrophil suspension.
[0020] 2) Culture in a cell culture incubator at 37°C and 5% CO2 for 12 hours.
[0021] 3) After the culture is completed, the cells are transferred to a 96-well round-bottom plate.
[0022] 3. Immunofluorescence detection of NETs produced by neutrophils after combined cytokine treatment:
[0023] 1) Treat neutrophils according to 2., centrifuge at 1800 rpm for 5 min, and discard the supernatant.
[0024] 2) Add 200 μL of 4% paraformaldehyde to each well and fix at room temperature for 10 min.
[0025] 3) Discard the 4% paraformaldehyde and wash three times with ice-cold PBS solution.
[0026] 4) Add 200 μL of 10% goat serum to each well and block for 1 hour at room temperature.
[0027] 5) Add 200 μL of primary antibody suspension (NETs-specific markers CitH3 and MPO) to each well.
[0028] 6) Incubate at 4°C overnight.
[0029] 7) The next day, discard the primary antibody and wash three times with PBS solution.
[0030] 8) Add 200 μL of secondary antibody suspension to each well and incubate at room temperature in the dark for 1 h.
[0031] 9) Discard the secondary antibody, add DAPI and seal the slide.
[0032] 10) Observe the co-localized fluorescence imaging under a confocal microscope.
[0033] 4. Enzyme-linked immunosorbent assay (ELISA) of neutrophil production of NETs:
[0034] 1) Extract and culture cells according to 2. After the culture, transfer the cells to a 96-well round-bottom plate, centrifuge at 1000g for 20 min, and collect the cell supernatant.
[0035] 2) Add 50 μL of sample and 100 μL of detection antibody labeled with horseradish peroxidase (HRP) to each well, seal the reaction wells with a sealing film, incubate in a 37°C incubator for 60 min, and let stand.
[0036] 3) Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350 μL), let stand for 1 min, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 5 times.
[0037] 4) Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 min.
[0038] 5) Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 min. BRIEF DESCRIPTION OF THE DRAWINGS
[0039] Figure 1 The purity chart of neutrophils prepared in Example 1 of the present invention.
[0040] FIG. 2 is a confocal micrograph of NETs obtained in Embodiment 3 of the present invention after fluorescence staining.
[0041] Figure 3 A quantitative statistical graph of NETs-specific markers obtained in embodiment 4 of the present invention. DETAILED DESCRIPTION
[0042] The instruments used in the following embodiments of the present invention are as follows:
[0043] Flow cytometer (Beckman, USA), multi-function microplate reader (Thermo Scientific, USA), laser scanning confocal microscope (Leica, Germany)
[0044] The specific embodiments of the present invention are further described in detail as follows:
[0045] Example 1: Method for determining the purity of neutrophils, the specific steps are as follows:
[0046] 1) Collect the newly purified neutrophils and transfer them to a 96-well round-bottom plate.
[0047] 2) Centrifuge at 1800 rpm for 5 min.
[0048] 3) Discard the supernatant and add 50 μL staining buffer and 0.5 μL PE-cy7-CD45, 0.5 μL FITC-CD66b and 0.5 μL Percp-cy5.5-CD16 to each well to resuspend the cells.
[0049] 4) Place in a 4°C refrigerator and stain for 30 minutes away from light.
[0050] 5) Add 100 μL staining buffer to each tube and centrifuge at 1800 rpm for 5 min.
[0051] 6) Discard the supernatant, add 150 μL PBS to resuspend the cells, filter out impurities with a cell strainer, and then detect on a flow cytometer.
[0052] Example 2: Inducing neutrophils in vitro by combined use of cytokines, the specific steps are as follows:
[0053] 1) According to Example 1, the neutrophils extracted were resuspended in a certain amount of RPMI 1640 complete medium, and the cell density was adjusted to 5×10 5 The number of holes.
[0054] 2) Add 10 ng / mL IFN-γ, 1 ng / mL GM-CSF and 30 μg / mL IFN-γ to each well of 200 μL cell suspension.
[0055] ng / mL IL-1β was used to induce neutrophils. A control group and an experimental group were set up in the plate. Mixed cytokines were added to the experimental group, and an equal volume of culture medium was added to the control group. Three replicate wells were set up for each group of samples.
[0056] 3) The treated cells were cultured in a 37°C, 5% CO2 incubator for 12 h.
[0057] 4) Collect the cells and transfer them into a 96-well round-bottom plate.
[0058] 5) Centrifuge at 1800 rpm for 5 min, discard the supernatant, and perform immunofluorescence staining.
[0059] Example 3: A method for inducing neutrophils to produce NETs in vitro, the specific steps are as follows:
[0060] 1) According to Example 1, the neutrophils extracted were resuspended in a certain amount of RPMI 1640 complete medium, and the cell density was adjusted to 5×10 5 The number of holes.
[0061] 2) A control group and an experimental group were set up in the plate. In the experimental group, 10 ng / mL IFN-γ, 1 ng / mL GM-CSF and 30 ng / mL IL-1β were added to 200 μL of cell suspension in each well to induce neutrophils, and an equal volume of culture medium was added to the control group. Three replicate wells were set up for each group of samples.
[0062] 3) The treated cells were cultured in a 37°C, 5% CO2 incubator for 12 h.
[0063] 4) Discard the supernatant of the collected cells and centrifuge at 1800 rpm for 5 min.
[0064] 5) Add 200 μL of 4% paraformaldehyde to each well and fix at room temperature for 10 min.
[0065] 6) Centrifuge at 1800 rpm for 5 min and discard the 4% paraformaldehyde.
[0066] 7) Add 200 μL of ice-cold PBS to each well and gently resuspend. Centrifuge at 1800 rpm for 5 min and discard the supernatant.
[0067] 8) Repeat 7) three times.
[0068] 9) Add 200 μL of 10% goat serum to each well and block for 1 hour at room temperature.
[0069] 10) Dilute the primary antibody mixture using immunostaining primary antibody diluent and add 200 μL of primary antibody mixture to each well.
[0070] The dilution ratio of CitH3 and MPO was 1:200 and incubated in a 4°C refrigerator overnight (>12h).
[0071] 11) The next day, place the cells in a 37°C incubator to rewarm for 30 minutes.
[0072] 12) Centrifuge at 1800 rpm for 5 min and discard the primary antibody.
[0073] 13) Add 200 μL PBS buffer solution to each well and gently resuspend. Centrifuge at 1800 rpm for 5 min and discard the supernatant.
[0074] 14) Repeat 13) three times.
[0075] 15) Dilute the secondary antibody mixture with 1% BSA in PBS solution, add 200 μL of the secondary antibody mixture to each well, and incubate the cells at room temperature in the dark for 1 hour. The secondary antibody dilution ratio is: CoraLite 488 (1:500), CoraLite
[0076] 594(1:200).
[0077] 16) Centrifuge at 1800 rpm for 5 min and discard the secondary antibody.
[0078] 17) Add 200 μL PBS buffer solution and gently resuspend, centrifuge at 1800 rpm for 5 min, and discard the supernatant.
[0079] 18) Repeat 17) three times.
[0080] 19) Pipette the cell suspension at the bottom of the 96-well round plate onto a glass slide, drop DAPI containing a fluorescence attenuation quencher onto the glass slide, and observe the fluorescence imaging under a confocal microscope.
[0081] Example 4: A method for identifying the content of NETs produced by neutrophils induced in vitro, the specific steps are as follows:
[0082] 1) Extract and culture cells according to 2. After the culture, transfer the cells to a 96-well round-bottom plate, centrifuge at 1000 g for 20 min, and collect the cell supernatant.
[0083] 2) Set up standard wells and sample wells, add 50 μL of standards of different concentrations to the standard wells, add 50 μL of the sample to be tested to the sample wells, and do not add anything to the blank wells.
[0084] 3) Except for the blank wells, add 100 μL of HRP-labeled detection antibody to each well of the standard wells and sample wells, seal the reaction wells with a sealing film, and incubate in a 37°C incubator for 60 min.
[0085] 4) Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350 μL), let stand for 1 min, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 5 times.
[0086] 5) Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 min.
[0087] 6) Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 min.
[0088] Figure 1 This is the purity of neutrophils after separation and purification, indicating that high-purity neutrophils were successfully separated.
[0089] FIG2 is a confocal micrograph of colocalization of NETs after fluorescent staining produced by the combined use of cytokines, indicating that the combined use of cytokines can induce neutrophils to produce NETs.
[0090] Figure 3 The quantitative analysis of NETs-specific markers produced by neutrophils indicated that the combined use of cytokines could increase the content of NETs.
Claims
1. A method for inducing neutrophils to produce NETs, comprising the steps of: (1) Isolation and purification of neutrophils; (2) The induction mode of NETs; (3) Quantitative analysis and identification of NETs.
2. The method according to claim 1, wherein the specific steps of separating and purifying neutrophils are as follows: at room temperature, a human peripheral blood sample is centrifuged using Ficoll density gradient centrifugation, and red blood cells are lysed to obtain purified CD45 + CD66b + CD16 + Neutrophils, purity over 95%.
3. According to the method of claim 1, the specific steps of inducing NETs are: treating neutrophils with 10ng / mL IFN-γ+1ng / mL GM-CSF+30ng / mL IL-1β for 12h to induce neutrophils to produce NETs.
4. According to the method of claim 1, the specific method for quantitative analysis and identification of NETs is: detecting NETs markers CitH3 and MPO by immunofluorescence, and analyzing their mean fluorescence intensity using ImageJ; quantifying NETs by detecting MPO-DNA and CitH3 in neutrophil supernatant by ELISA.