Preparation method and application of neutrophil extracellular capture net positive cells

Abstract

The invention discloses a preparation method and application of neutrophil extracellular capture net positive cells. The preparation method comprises the following steps: dissolving sodium bicarbonate, ammonium chloride and disodium ethylene diamine tetraacetate with purified water to prepare a stimulation solution; in the stimulation liquid, the concentration of sodium bicarbonate is 0.8-1g / L, the concentration of ammonium chloride is 8-10g / L, and the concentration of disodium ethylene diamine tetraacetate is 0.05-0.1 g / L; jointly incubating the blood and the stimulation liquid for 30-60 minutes, centrifuging, and discarding the supernatant; and re-suspending the cells by using a phosphate buffer solution, centrifuging, and discarding the supernatant to obtain the neutrophil extracellular capture net positive cells. According to the method, cell culture work and sterile cell operation are not needed, the overall operation time can be shortened to be within 1-1.5 hours, and compared with an existing method, the time is greatly saved, the technical requirements for operators are reduced, and meanwhile the requirements for instruments and equipment are reduced.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a preparation method and application of neutrophil extracellular capture net-positive cells. Background Art

[0002] Neutrophils are one of the main responders of acute inflammation, accounting for 50% to 70% of all circulating leukocytes in humans, and are the initial defense against invading pathogens. During infection, neutrophils migrate from peripheral blood to tissues, where they can rapidly kill pathogens after phagocytosis, but can also maximize the host immune response by releasing their powerful antimicrobial arsenal, including granzymes, reactive oxygen species, and neutrophil extracellular traps (NETs).

[0003] NETs are secreted by activated neutrophils and are composed of a mesh structure of DNA, histones, and antimicrobial proteins that are responsible for capturing and killing extracellular pathogens. The process of NET formation is called neutrophil extracellular trapping NETosis, which is an important immune response that helps defend the body from microorganisms. However, excessive or inappropriate NETosis may be associated with the occurrence and development of some diseases, including autoimmune diseases, infections, inflammatory diseases, etc. Therefore, the study of extracellular trapping NETosis helps to gain a deeper understanding of the function of the immune system and its role in disease.

[0004] In the actual testing of detection technology, in order to determine the effectiveness of the detection reagent, NETs-positive cells need to be used for product quality control. The existing preparation of NETs-positive cells is mainly through extracting neutrophils from the blood, and stimulating them with phorbol ester (PMA) and culturing them for more than 4 hours to obtain NETs-positive cells. The overall operation time exceeds 6 hours, and professional cell culture equipment and cell culture environment are required to prepare positive cells.

[0005] Therefore, there is an urgent need for a method that can quickly and effectively prepare NETs-positive cells. Summary of the invention

[0006] In order to solve the above technical problems, the purpose of the present invention is to provide a preparation method and application of neutrophil extracellular capture network-positive cells; this preparation method does not require cell culture work, does not require sterile cell operation, and the overall operation time can be shortened to within 1 to 1.5 hours. Compared with the existing methods, it greatly saves time and reduces the technical requirements for operators, while reducing the requirements for instruments and equipment.

[0007] In order to achieve the above technical objectives and the above technical effects, the present invention is implemented through the following technical solutions:

[0008] In one aspect, the present invention provides a method for preparing neutrophil extracellular capture network-positive cells, comprising the following steps:

[0009] S1, taking sodium bicarbonate, ammonium chloride, and disodium ethylenediaminetetraacetate, dissolving them in purified water, and preparing a stimulation solution; in the stimulation solution, the concentration of sodium bicarbonate is 0.8-1 g / L, the concentration of ammonium chloride is 8-10 g / L, and the concentration of disodium ethylenediaminetetraacetate is 0.05-0.1 g / L;

[0010] S2, incubating the blood with the stimulation solution prepared in step S1 for 30 to 60 minutes, centrifuging, and discarding the supernatant;

[0011] S3, resuspend the cells in phosphate buffer, centrifuge, discard the supernatant, and obtain neutrophil extracellular trap-positive cells.

[0012] Furthermore, in step S1, the pH of the stimulation solution is 7.2-7.4.

[0013] Furthermore, in step S2, the volume ratio of blood to stimulation fluid is (1-1.5) mL: (40-45) mL.

[0014] Furthermore, in step S2, the incubation process is performed in multiple times.

[0015] Furthermore, the incubation process in step S2 is preferably the following process:

[0016] Incubate the blood with the stimulation solution for 5 minutes, centrifuge at 1000-2000rpm for 5 minutes, and discard the supernatant; resuspend the cells with the stimulation solution again, incubate for 5 minutes, centrifuge at 1000-2000rpm for 5 minutes, and discard the supernatant; resuspend the cells with the stimulation solution for the third time, incubate for 20 minutes, centrifuge at 1000-2000rpm for 5 minutes, and discard the supernatant.

[0017] Furthermore, in step S3, the time for resuspending the cells using phosphate buffer is 2 to 20 minutes.

[0018] Furthermore, in step S3, the cells are resuspended in phosphate buffer for multiple times; specifically, the cells are resuspended in phosphate buffer and then centrifuged to discard the supernatant; after repeating this step 1 to 2 times, the cells are resuspended in phosphate buffer containing 4% paraformaldehyde, fixed for 15 minutes, and then centrifuged to discard the supernatant.

[0019] Furthermore, the neutrophil extracellular trap-positive cells are preserved in the following manner: the cells are resuspended in phosphate buffer and stored at 4-6°C.

[0020] Another aspect of the present invention provides an application of the neutrophil extracellular trapping network-positive cells, specifically: the neutrophil extracellular trapping network-positive cells can be used as a positive control substance for NETs detection reagents.

[0021] The beneficial effects of the present invention are:

[0022] The present invention adopts a stimulation solution-induced incubation of blood cells that is different from the prior art to obtain neutrophil extracellular capture network-positive cells; the method of the present invention does not require cell culture work or sterile cell operation, and the overall operation time can be shortened to within 1 to 1.5 hours. Compared with the existing methods, it greatly saves time and reduces the technical requirements for operators, while reducing the requirements for instruments and equipment.

[0023] The neutrophil extracellular capture net-positive cells prepared by the present invention can be used as positive control substances for NETs detection reagents (such as MPO detection reagents and / or citH3 detection reagents). BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of neutrophil flow cytometry grouping of sample 1 in Example 1 of the present invention, wherein neutrophils are distinguished by setting a gate based on CD66c; wherein (1A) is a schematic diagram of neutrophil flow cytometry grouping of blood sample 1 that has not been treated by the method of the present invention; and (2B) is a schematic diagram of neutrophil flow cytometry grouping of blood sample 1 that has been treated by the method of the present invention;

[0025] Figure 2 Schematic diagram of neutrophil flow cytometric grouping of sample 1 in Example 2 of the present invention, wherein neutrophils are distinguished by setting a gate based on CD66c; wherein (2A) is a schematic diagram of neutrophil flow cytometric grouping of blood sample 1 that has not been treated by the method of the present invention; and (2B) is a schematic diagram of neutrophil flow cytometric grouping of blood sample 1 that has been treated by the method of the present invention. DETAILED DESCRIPTION

[0026] The technical solutions in the present invention will be described clearly and completely below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] The present invention provides a method for preparing neutrophil extracellular capture net-positive cells, comprising the following steps:

[0028] S1, taking sodium bicarbonate, ammonium chloride, and disodium ethylenediaminetetraacetate, dissolving them in purified water, and preparing a stimulation solution; in the stimulation solution, the concentration of sodium bicarbonate is 0.8-1 g / L, the concentration of ammonium chloride is 8-10 g / L, and the concentration of disodium ethylenediaminetetraacetate is 0.05-0.1 g / L; the pH of the stimulation solution is 7.2-7.4;

[0029] S2, incubating the blood with the stimulation solution prepared in step S1 for 30 to 60 minutes, centrifuging, and discarding the supernatant;

[0030] The volume ratio of blood to stimulation solution is (1-1.5) mL: (40-45) mL;

[0031] The incubation process is carried out in multiple steps, preferably in the following steps:

[0032] The blood was incubated with the stimulation solution for 5 minutes, centrifuged at 1000-2000 rpm for 5 minutes, and the supernatant was discarded; the cells were resuspended with the stimulation solution again, incubated for 5 minutes, centrifuged at 1000-2000 rpm for 5 minutes, and the supernatant was discarded; the cells were resuspended with the stimulation solution for the third time, incubated for 20 minutes, centrifuged at 1000-2000 rpm for 5 minutes, and the supernatant was discarded;

[0033] S3, resuspend the cells in phosphate buffer, centrifuge, discard the supernatant, and obtain neutrophil extracellular trap-positive cells;

[0034] In step S3, the time for resuspending the cells using phosphate buffer is 2 to 20 minutes.

[0035] In step S3, the cells are resuspended in phosphate buffer for multiple times; specifically, the cells are resuspended in phosphate buffer and then centrifuged, and the supernatant is discarded; after repeating this step 1 to 2 times, the cells are resuspended in phosphate buffer containing 4% paraformaldehyde, fixed for 15 minutes, and then centrifuged and the supernatant is discarded. The process of resuspending the cells in phosphate buffer can wash away lysed cell fragments, such as red blood cell lysed fragments.

[0036] The neutrophil extracellular trap-positive cells are preserved in the following manner: the cells are resuspended in a phosphate buffer solution and stored at 4-6°C.

[0037] The present invention also provides an application of the neutrophil extracellular trapping network-positive cells, specifically: the neutrophil extracellular trapping network-positive cells can be used as a positive control substance for NETs detection reagents (such as MPO detection reagents and / or citH3 detection reagents).

[0038] The present invention is further described in detail below through specific examples.

[0039] Example 1

[0040] S1, prepare stimulation solution; the stimulation solution formula is as follows: 0.84 g / L sodium bicarbonate, 8.28 g / L ammonium chloride, 0.075 g / L disodium ethylenediaminetetraacetic acid, after dissolving with purified water, adjust the pH to 7.3;

[0041] S2, take fresh blood (blood collection should not exceed 2 hours), incubate 1 mL of blood with 40 mL of stimulation solution for 5 minutes; centrifuge at 1500 rpm for 5 minutes, and discard the supernatant; resuspend the cells with the same amount of stimulation solution as the first time, incubate for 5 minutes, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant; resuspend the cells with the same amount of stimulation solution as the first time for the third time, incubate for 20 minutes, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant;

[0042] S3, resuspend the cells with 1 mL phosphate buffered saline (PBS), centrifuge at 1500 rpm for 5 minutes, and discard the supernatant; repeat this step twice; resuspend the cells with phosphate buffered saline (PBS) containing 4% paraformaldehyde, fix for 15 minutes, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant;

[0043] S4, resuspend the cells in 1 mL phosphate-buffered saline (PBS) and store at 4°C.

[0044] Ten blood samples were taken and processed according to the above steps respectively; the main indicator of NETs, ​​myeloperoxidase (MPO), was used to detect the ten blood samples that were not treated by the method of the present invention and were treated by the method of the present invention using a myeloperoxidase (MPO) detection reagent. The test results are shown in Table 1. The cell flow cytometry clustering diagram of sample 1 is shown in Figure 1 As shown, there is a significant difference between the positive rate of blood samples not treated by the method of the present invention and the positive rate of blood samples treated by the method of the present invention.

[0045] Table 1

[0046]

[0047]

[0048] It can be seen from this that the neutrophil extracellular capture network-positive cells prepared by the method of the present invention can be used as a positive control substance for myeloperoxidase (MPO) detection reagent.

[0049] Example 2

[0050] S1, prepare stimulation solution; the stimulation solution formula is as follows: 0.85 g / L sodium bicarbonate, 8.5 g / L ammonium chloride, 0.085 g / L disodium ethylenediaminetetraacetic acid, after dissolving with purified water, adjust the pH to 7.2;

[0051] S2, take fresh blood (blood collection should not exceed 2 hours), incubate 1 mL of blood with 40 mL of stimulation solution for 5 minutes; centrifuge at 1500 rpm for 5 minutes, and discard the supernatant; resuspend the cells with the same amount of stimulation solution as the first time, incubate for 5 minutes, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant; resuspend the cells with the same amount of stimulation solution as the first time for the third time, incubate for 25 minutes, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant;

[0052] S3, resuspend the cells with 1 mL phosphate buffered saline (PBS), centrifuge at 1500 rpm for 5 minutes, and discard the supernatant; repeat this step twice; resuspend the cells with phosphate buffered saline (PBS) containing 4% paraformaldehyde, fix for 15 minutes, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant;

[0053] S4, resuspend the cells in 1 mL phosphate-buffered saline (PBS) and store at 4°C.

[0054] Ten blood samples were taken and processed according to the above steps respectively; the main indicator of NETs, ​​citrullinated histone H3 (CitH3), was used to detect the ten blood samples that were not treated by the method of the present invention and were treated by the method of the present invention using the CitH3 detection reagent. The test results are shown in Table 2. The cell flow cytometry clustering diagram of sample 1 is shown in Figure 2 As shown, there is a significant difference between the positive rate of blood samples not treated by the method of the present invention and the positive rate of blood samples treated by the method of the present invention.

[0055] Table 2

[0056]

[0057]

[0058] It can be seen that the neutrophil extracellular trap-positive cells prepared by the method of the present invention can be used as a positive control for citrullinated histone H3 (CitH3) detection reagents.

[0059] Compared with the prior art, the advantages of the present invention are shown in Table 3 below.

[0060] Table 3

[0061]

[0062] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0063] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for preparing neutrophil extracellular trap-positive cells, characterized in that: The steps include: S1, taking sodium bicarbonate, ammonium chloride, and disodium ethylenediaminetetraacetate, dissolving them in purified water, and preparing a stimulation solution; in the stimulation solution, the concentration of sodium bicarbonate is 0.8-1 g / L, the concentration of ammonium chloride is 8-10 g / L, and the concentration of disodium ethylenediaminetetraacetate is 0.05-0.1 g / L; S2, incubating the blood with the stimulation solution prepared in step S1 for 30 to 60 minutes, centrifuging, and discarding the supernatant; S3, resuspend the cells in phosphate buffer, centrifuge, discard the supernatant, and obtain neutrophil extracellular trap-positive cells.

2. The method for preparing neutrophil extracellular trap-positive cells according to claim 1, characterized in that: In step S1, the pH of the stimulation solution is 7.2-7.

4.

3. The method for preparing neutrophil extracellular trap-positive cells according to claim 1, characterized in that: In step S2, the volume ratio of blood to stimulation solution is (1-1.5) mL: (40-45) mL.

4. The method for preparing neutrophil extracellular trap-positive cells according to claim 1, characterized in that: In step S2, the incubation process is performed in multiple times.

5. The method for preparing neutrophil extracellular trap-positive cells according to claim 1, characterized in that: The incubation process in step S2 is: Incubate the blood with the stimulation solution for 5 minutes, centrifuge at 1000-2000rpm for 5 minutes, and discard the supernatant; resuspend the cells with the stimulation solution again, incubate for 5 minutes, centrifuge at 1000-2000rpm for 5 minutes, and discard the supernatant; resuspend the cells with the stimulation solution for the third time, incubate for 20 minutes, centrifuge at 1000-2000rpm for 5 minutes, and discard the supernatant.

6. The method for preparing neutrophil extracellular trap-positive cells according to claim 1, characterized in that: In step S3, the time for resuspending the cells in phosphate buffer is 2 to 20 minutes.

7. The method for preparing neutrophil extracellular trap-positive cells according to claim 6, characterized in that: In step S3, the cells are resuspended in phosphate buffer for multiple times; specifically, the cells are resuspended in phosphate buffer and then centrifuged to discard the supernatant; after repeating this step 1 to 2 times, the cells are resuspended in phosphate buffer containing 4% paraformaldehyde, fixed for 15 minutes, and then centrifuged to discard the supernatant.

8. The method for preparing neutrophil extracellular trap-positive cells according to claim 1, characterized in that: Neutrophil extracellular trap-positive cells were preserved in the following manner: cells were resuspended in phosphate buffered saline and stored at 4-6°C.

9. The use of the neutrophil extracellular capture net-positive cells according to any one of claims 1 to 8, characterized in that: The neutrophil extracellular trapping network-positive cells can be used as positive controls for NETs detection reagents.

Images