Mouse neutrophil magnetic bead sorting product and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a mouse neutrophil magnetic bead sorting product and application. The invention provides a product for mouse neutrophil negative sorting and a corresponding sorting method. The sorted product can be used for sorting bone marrow, spleen and peripheral blood neutrophil. The sorting steps are simple, the time is short, and the sorted neutrophil is high in purity and good in activity and has a relatively good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a mouse neutrophil magnetic bead sorting product and application. Background Art

[0002] Neutrophils are one of the most important innate immune cells in the mammalian immune system. They play a key role in defending against pathogen infection, regulating inflammatory responses, and clearing cellular debris. Due to their central role in immune responses and disease, obtaining highly pure and active neutrophils is extremely important for studying their biological functions.

[0003] Currently, a variety of methods are available for isolating mouse neutrophils, but traditional separation techniques still suffer from drawbacks such as cumbersome procedures, long time consumption, limited application scope, and unstable sorting results. Therefore, the development of a rapid and efficient mouse neutrophil sorting product is particularly important. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a mouse neutrophil magnetic bead sorting product and application.

[0005] The present invention provides a composition of biotin-labeled antibodies, wherein the antibodies include: CD4, CD5, bCD19, CD16 / 32, CD117 and bAB20.

[0006] Furthermore, the concentration of the biotin-labeled CD4 is 0.1 mg / mL to 1 mg / mL; the concentration of the biotin-labeled CD5 is 0.1 mg / mL to 1 mg / mL; the concentration of the biotin-labeled bCD19 is 0.1 mg / mL to 1 mg / mL; the concentration of the biotin-labeled CD16 / 32 is 0.1 mg / mL to 1 mg / mL; the concentration of the biotin-labeled CD117 is 0.1 mg / mL to 1 mg / mL; and the concentration of the biotin-labeled bAB20 is 0.1 mg / mL to 1 mg / mL. By adjusting the concentrations and ratios used, in a specific embodiment of the present invention, a good sorting effect can be achieved when the concentration of each biotin-labeled antibody component is 0.5 mg / mL.

[0007] The present invention provides a reagent comprising the composition of the present invention and auxiliary materials.

[0008] Furthermore, the excipient is used to maintain the activity of the composition or assist it in functioning; in a specific embodiment of the present invention, the excipient includes at least one of a preservative, an antibody stabilizer and / or a buffer.

[0009] The present invention provides a kit comprising at least one of the composition of the present invention and / or the reagents of the present invention, a detergent and / or a sorting buffer.

[0010] Furthermore, the separation buffer is selected from at least one of the following:

[0011] PBS containing EDTA and fetal bovine serum (FBS); and / or

[0012] PBS containing EDTA and bovine serum albumin (BSA);

[0013] Furthermore, the separation buffer is selected from at least one of the following:

[0014] PBS containing 1-3 mM EDTA and 1%-3% fetal bovine serum;

[0015] PBS containing 1–3 mM EDTA and 0.1 wt%–1 wt% bovine serum albumin (BSA);

[0016] Specifically, the separation buffer is selected from at least one of the following:

[0017] PBS containing 2 mM EDTA and 2% vt fetal bovine serum;

[0018] PBS containing 2mM EDTA and 0.5%wt bovine serum albumin, wherein PBS does not contain calcium and magnesium ions.

[0019] The terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, e.g., not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0020] The use of any and all examples or exemplary language, such as "such as" or "including," herein is intended merely to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0021] The present invention provides the use of any one of the following I) to III) in neutrophil sorting:

[0022] 1), the composition of the present invention;

[0023] II), the reagent of the present invention;

[0024] III), the kit of the present invention.

[0025] The present invention provides a method for sorting neutrophils, characterized by comprising sorting neutrophils in a sample using at least one of the following i) to iii):

[0026] i), the composition of the present invention;

[0027] ii), the reagent of the present invention;

[0028] iii), the kit of the present invention.

[0029] Furthermore, the method of the present invention comprises the following steps:

[0030] Step 1: mixing the sample with the composition of the present invention and performing a first incubation;

[0031] Step 2, the product after the first incubation is incubated with streptavidin-labeled magnetic beads for a second time;

[0032] Step 3: performing magnetic separation on the product after the second incubation to obtain the neutrophils.

[0033] Further,

[0034] The concentration of cells in the sample was 1×10 8 / mL;

[0035] The first incubation condition is 2°C to 6°C for 5 min to 15 min;

[0036] The second incubation condition is 2°C to 6°C for 5 min to 15 min;

[0037] The magnetic separation time is 2 to 6 minutes;

[0038] After the first incubation in step 1 and before step 2, the steps of washing and centrifugation are further included.

[0039] In a specific embodiment of the present invention, the method for separating neutrophils comprises the following steps:

[0040] Step 1: mixing the sample with the composition of the present invention and performing a first incubation;

[0041] Step 2, washing and centrifuging the product after the first incubation;

[0042] Step 3, the centrifuged product is incubated with streptavidin-labeled magnetic beads for a second time;

[0043] Step 4: Perform magnetic separation on the product after the second incubation.

[0044] More specifically:

[0045] Step 1: Resuspend the lysed bone marrow, peripheral blood or spleen cells in the sorting buffer and adjust the cell density to 1×10 8 / mL, obtain the sample.

[0046] Step 2, incubating the sample and the composition for 10 minutes;

[0047] Step 3: After incubation, add sorting buffer for washing, centrifuge and resuspend in sorting buffer.

[0048] Step 4: The reselected product was incubated with streptavidin-labeled magnetic beads for 10 min;

[0049] Step 5: Add sorting buffer, resuspend, and magnetically absorb for 5 minutes;

[0050] Step 6: Centrifuge and collect the product after magnetic separation to obtain the neutrophils.

[0051] In the method of the present invention, the sample includes bone marrow cells, spleen cells and / or peripheral blood.

[0052] The present invention optimizes a composition of biotin-labeled antibodies, including screening of antibody combinations and ratios. Test results show that a composition of biotin-labeled antibodies composed of the six antibodies described in the present invention has the best detection effect. Furthermore, on this basis, the dosage of the antibodies is optimized. After optimization, the detection results are accurate and the detection efficiency is high. In addition, the detection steps are adjusted to further improve the accuracy of the detection. Therefore, in the present invention, the various parameter characteristics involved influence each other and jointly affect the detection results, and should be protected as a whole.

[0053] In addition, the sorting method of the present invention does not require a separation column and is simple. Compared with the mouse neutrophil sorting product (cat. no. 19762) launched by STEMCELL, the present invention has superior purity (p < 0.01) while ensuring comparable sorted cell viability, and has a significant advantage in sorting time.

[0054] The present invention provides a product for negatively sorting mouse neutrophils and a corresponding sorting method. The sorting product of the present invention can be used to sort neutrophils from mouse bone marrow, spleen, and peripheral blood. The sorting process is simple, the time is short, and the sorted neutrophils have high purity and good activity, and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The figure shows the sorting of neutrophils from bone marrow of C57BL / 6 mice;

[0056] Figure 2 The figure shows the sorting of neutrophils from the bone marrow of BALB / C mice;

[0057] Figure 3 Shows the sorting of neutrophils from the spleen of C57BL / 6 mice;

[0058] Figure 4 Shows the sorting of neutrophils from the spleen of BALB / C mice;

[0059] Figure 5 The figure shows the separation of mouse peripheral blood neutrophils;

[0060] Figure 6 Shows the cell viability after sorting;

[0061] Figure 7 The figure shows the ability of sorted neutrophils to produce ROS under PMA stimulation;

[0062] Figure 8 Figure 2 shows representative results of flow cytometry staining of neutrophil Ly6G and CD11b obtained after optimizing the sorting process (after optimization, the cells were washed);

[0063] Figure 9 Figure 2 shows an experiment to optimize antibody dosage; A shows the statistical analysis of cell purity after sorting neutrophils from C57BL / 6 mouse bone marrow using 0.5 µL, 1.5 µL, and 2.5 µL of antibody (n=4); B shows the statistical analysis of cell purity after sorting BALB / C neutrophils using 1 µL, 1.5 µL, and 2 µL of antibody (n=3) after further optimizing antibody dosage.

[0064] Figure 10 Figure 2 shows a comparison of the purity of bone marrow neutrophils obtained by sorting using the present invention and the STEMCELL product (cat. no. 19762); A is a representative result of flow cytometry staining of Ly6G and CD11b in sorted cells; B is a statistical analysis of purity; **P<0.01, n=3;

[0065] Figure 11 Comparison of the purity of bone marrow neutrophils obtained by the present invention and the STEMCELL product (cat. no. 19762) within the time limit; A is a representative result of flow cytometric staining of Ly6G and CD11b cells after sorting; B is a statistical analysis of purity; **P<0.01, n=3;

[0066] Figure 12 Comparison of bone marrow neutrophil viability obtained by sorting using the present invention and the STEMCELL product (cat. no. 19762), and representative results of 7AAD flow cytometry staining. DETAILED DESCRIPTION

[0067] The present invention provides mouse neutrophil magnetic bead sorting products and applications. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0068] In addition to the six antibodies and their ratios in the currently described biotin-antibody mix, other antibodies capable of recognizing non-target cells can also be used, or existing antibodies can be optimized and improved, such as by slightly adjusting the antibody ratio or adding other biotin-labeled antibodies. Such improved antibody combinations can still achieve the same sorting objectives as the prior art and should be considered within the scope of protection of this invention.

[0069] The present invention uses 300nm SA magnetic beads, however, alternative magnetic bead specifications can also achieve similar effects. For example, magnetic beads with a diameter of 50nm~5μm can be used, or other types of magnetic particle carriers can be used. When combined with the corresponding streptavidin protein binding mechanism, efficient neutrophil sorting can still be achieved. These are all based on the premise of the principles of the present invention, and several improvements can be made, which should also be considered as the scope of protection of the present invention.

[0070] The steps of the present invention are designed to incubate the antibody first, wash, and then add SA magnetic beads. However, simplification of the steps can be considered, for example, directly incubating with the magnetic beads after the antibody incubation is completed. This can reduce the number of steps and shorten the sorting time. In addition, the incubation time of the magnetic beads can be adjusted between 5 and 20 minutes according to the experimental requirements, and similar sorting effects can still be achieved. These adjustments are all within the scope of protection of the present invention.

[0071] Compared with human neutrophils, mouse neutrophils account for a lower proportion in peripheral blood, are more difficult to sort, and are easily confused with other cell types.

[0072] The reagents and consumables used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below with reference to the following examples:

[0073] Example 1 Mouse Neutrophil Magnetic Bead Sorting Product and Application

[0074] 1. Preparation of Reagents, Antibodies, and Cell Samples

[0075] 1. Using Beaver Bioengineering's patented protein coupling technology, streptavidin (SA) is covalently linked to the surface of 300 nm carboxyl magnetic beads (cat. no. 22308C), enabling efficient binding of biotinylated antibodies. The beads are superparamagnetic microspheres with uniform particle size and regular morphology, facilitating the convenient and rapid capture of target cells.

[0076] 2. Use Biotin-labeled antibodies from Beaver Bioengineering Co., Ltd. and adjust the antibody dosage ratio to obtain a Biotin-antibody Mix. The concentration of each antibody component of the Biotin-antibody Mix is ​​0.5 mg / mL to effectively label non-neutrophils.

[0077] 3. Cell sample preparation

[0078] (1) Take the mouse bone marrow, spleen or peripheral blood cells and place them in a centrifuge tube for red blood cell lysis.

[0079] (2) After lysis, resuspend the cells in PBS, filter the cell suspension through a 70 μm cell sieve, and centrifuge at 500 g for 5 minutes.

[0080] Note: The cell suspension needs to be passed through a cell sieve to remove tissue and cell clumps, otherwise it will affect the purity of subsequent cell sorting.

[0081] (3) After centrifugation, resuspend the cells in sorting buffer, wash the cells, centrifuge at 500g for 5 minutes, and discard the supernatant.

[0082] Note: The sorting buffer is PBS containing 2mM EDTA and 2wt% fetal bovine serum (FBS) or PBS containing 2mM EDTA and 0.5wt% BSA, which must be sterilized by filtration through a 0.22μm filter membrane in advance.

[0083] 3.4 Resuspend the cells in sorting buffer and adjust the cell density to 1×10 8 cells / mL.

[0084] 2. Sorting process

[0085] 1. Add 100 μL cell suspension (1×10 7 Add 2 μL of Biotin-Antibody mixture to the bottom of a 1.5 mL centrifuge tube (for example, 10 cells). Mix well and incubate at 4°C for 10 min.

[0086] Note: When adding cell suspension, add cells to the bottom of the centrifuge tube and avoid adding along the tube wall. If more cells are to be sorted, increase the amount of Biotin-Antibody Mix proportionally. The amount of antibody has been optimized in this experiment, and the optimal antibody amount is currently used. Small adjustments within this range usually do not significantly affect the sorting effect ( Figure 9 ).

[0087] 2. After incubation, wash the cells once: add sorting buffer to 1.5 mL and centrifuge at 500 g for 5 minutes.

[0088] Note: If the number of cells to be sorted is too large, use a 15 mL centrifuge tube, add sorting buffer to 5-10 mL, and then centrifuge.

[0089] 3. After centrifugation, discard the supernatant and resuspend the cells in 100 μL sorting buffer.

[0090] Note: If more cells are to be sorted, the amount of sorting buffer should be increased proportionally. For example, if the amount before sorting is 2×10 7 If the cells are large, resuspend them in 200 μL sorting buffer.

[0091] 4. Add 20 μL of washed SA magnetic beads to the centrifuge tube (the magnetic beads need to be washed with sorting buffer before use: vortex and resuspend the magnetic beads, pipette the magnetic beads needed for the experiment into a 1.5 mL centrifuge tube, add 1 mL of sorting buffer, centrifuge at 10,000 g for 1 min, and discard the supernatant. Add 1 mL of sorting buffer to wash the magnetic beads once, and then resuspend the magnetic beads with the same volume of sorting buffer as the original. If 20 μL of magnetic beads are aspirated for washing, resuspend them with 20 μL of sorting buffer after washing), mix thoroughly, and incubate at 4°C for 10 min.

[0092] Note: If more cells are to be sorted, the amount of SA magnetic beads should be increased proportionally. For example, if 5×10 7 For cells, add 10 μL Biotin-Antibody Mix and 100 μL SA magnetic beads to 500 μL cell suspension. 7 If the number of cells is too high, the volume of the cell suspension is made up to 100 μL, and 2 μL of Biotin-Antibody Mix and 20 μL of SA magnetic beads are added.

[0093] 5. After incubation, transfer the tube to a flow cytometry tube, add selection buffer to 2.5 mL, and mix thoroughly with a pipette (avoid vigorous shaking or inversion).

[0094] 6. Place the cell separation tube on the magnetic rack and let it stand for 5 minutes.

[0095] 7. Gently pour the cell suspension into a sterile centrifuge tube (do not remove the tube from the magnetic stand during pouring). This cell suspension contains the purified mouse neutrophils. Centrifuge at 500g for 5 minutes. Discard the supernatant after centrifugation to collect the cells.

[0096] 8. After washing the cells according to experimental needs, resuspend the cells in the required buffer or culture medium and use them for subsequent molecular biology or cell biology experiments.

[0097] 3. Sorting effect

[0098] 1. Cells before and after sorting were labeled with FITC anti-mouse Ly-6G antibody (clone 1A8) and PE anti-mouse CD11b antibody (clone M1 / 70) and analyzed by flow cytometry;

[0099] Using mouse bone marrow cells, the purity of Ly6G+CD11b+ neutrophils before and after sorting was: 46.41% (C57BL / 6, before sorting), 93.23% (C57BL / 6, after sorting) ( Figure 1 ); 46.37% (BALB / C, before sorting), 95.04% (BALB / C, after sorting) ( Figure 2 ).

[0100] Using mouse spleen cells, the purity of Ly6G+CD11b+ neutrophils before and after sorting was: 2.99% (C57BL / 6, before sorting), 89.78% (C57BL / 6, after sorting) ( Figure 3 ); 6.30% (BALB / C, before sorting), 91.84% (BALB / C, after sorting) ( Figure 4 ).

[0101] Using mouse peripheral blood, the purity of Ly6G+ CD11b+ neutrophils before and after sorting were 20.04% and 93.82% (C57BL / 6) ( Figure 5 ).

[0102] 2. The sorted cells were stained with 7AAD, and the neutrophil viability reached 99% after sorting ( Figure 6 ).

[0103] 3. Functional testing of bone marrow neutrophils after sorting.

[0104] The neutrophils obtained by sorting the products involved in this invention were analyzed for their ability to produce ROS under PMA stimulation. PMA was used to stimulate the cells at 37°C for 10 minutes to induce ROS, and the oxidation of the substrate DCFH was detected by flow cytometry to evaluate the ability to generate ROS in the cells. The unstimulated samples served as blank controls in a resting state, while the samples treated with a reactive oxygen species positive treatment agent (Rosup, Yisheng ROS Detection Kit) served as positive controls. The results showed that after PMA stimulation, the level of ROS release from neutrophils increased significantly, indicating that these cells can be activated normally and are in good functional condition ( Figure 7 ).

[0105] The above data show that by applying this invention, high-purity neutrophils can be obtained without density gradient centrifugation, and the cells can maintain good biological functions.

[0106] Example 2 Optimization of Mouse Neutrophil Magnetic Bead Sorting Product

[0107] 1. The impact of washing

[0108] The results showed that the sorting effect was better when the antibody incubation was followed by washing and then incubation with magnetic beads (right: washing) ( Figure 8 ).

[0109] 2. Antibody dosage

[0110] The results showed that 2 μL of mixed antibody can achieve a good sorting effect. The concentration of each antibody component is 0.5 mg / mL under this dosage. Therefore, the present invention uses a smaller amount of antibody ( Figure 9 ).

[0111] Example 3 Comparison with existing products

[0112] Comparison of the purity of bone marrow neutrophils obtained by the present invention and STEMCELL product (cat. no. 19762) Figure 10 The present invention was significantly superior to the present invention and the STEMCELL product (P<0.01, n=3).

[0113] Comparison of the purity of bone marrow neutrophils obtained by the present invention and STEMCELL product (cat. no. 19762) within the time limit ( Figure 11 The present invention was significantly superior to the present invention and the STEMCELL product (P<0.01, n=3).

[0114] Comparison of the viability of bone marrow neutrophils obtained by the present invention and STEMCELL product (cat. no. 19762) ( Figure 12) Cell activity was comparable, but the present invention was slightly superior to the STEMCELL product. Representative results of 7AAD flow cytometry staining.

[0115] Through multiple parallel experiments. The experimental results show that under normal operating conditions, the operation time of the STEMCELL sorting product requires at least 53 minutes (see its product manual for detailed steps), while the present invention only requires 30 minutes; the comparative results show that the present invention achieves higher sorting purity in a shorter time while ensuring cell viability (94.25±0.79% vs. 87.58±1.55%, P<0.01). In addition, we further shortened the operation time to 15 minutes (the present invention) and 20 minutes (STEMCELL sorting product) for testing. The results show that even within a shorter operating time, the purity of neutrophils sorted by the present invention is still higher than that of the STEMCELL sorting product (74.89±1.01% vs. 67.66±2.27%, P<0.01). Therefore, while ensuring the purity and viability of sorted cells, the present invention has a significant time advantage ( Figures 10-12 ).

[0116] Specifically, the present invention uses the following steps to complete the sorting in 30 minutes:

[0117] 1. Add 100 μL of mouse bone marrow cell suspension (1×10 7 cells) were added to the bottom of a 1.5 mL centrifuge tube, and then 2 μL of Biotin-Antibody mixture was added, mixed and incubated at 4°C for 10 min.

[0118] 2. After incubation, wash the cells once, add sorting buffer to 1.5 mL, and centrifuge at 500 g for 5 min (sorting buffer is PBS containing 2 mM EDTA and 2% fetal bovine serum (FBS)).

[0119] 3. After centrifugation, discard the supernatant and resuspend the cells in 100 μL of sorting buffer.

[0120] 4. Add 20 μL of washed SA magnetic beads to the centrifuge tube (the beads were washed twice with sorting buffer before use and then resuspended in the same volume of sorting buffer as the original volume), mix thoroughly, and incubate at 4°C for 10 min.

[0121] 5. After incubation, transfer the cells to a flow cytometry tube, add separation buffer to 2.5 mL, and mix thoroughly by pipetting up and down.

[0122] 6. Place the cell separation tube on the magnetic stand and let it stand for 5 minutes.

[0123] 7. Gently pour the cell suspension into a sterile centrifuge tube. This cell suspension contains the purified mouse neutrophils.

[0124] The present invention adopts the following steps to complete the sorting in 15 minutes:

[0125] 1. Add 100 μL of mouse bone marrow cell suspension (1×10 7 cells) were added to the bottom of a 1.5 mL centrifuge tube, and then 2 μL of Biotin-Antibody mixture was added, mixed and incubated at 4°C for 5 min.

[0126] 2. After incubation, wash the cells once, add sorting buffer to 1.5 mL, and centrifuge at 500 g for 3 min (sorting buffer is PBS containing 2 mM EDTA and 2% fetal bovine serum (FBS)).

[0127] 3. After centrifugation, discard the supernatant and resuspend the cells in 100 μL of sorting buffer.

[0128] 4. Add 20 μL of washed SA magnetic beads to the centrifuge tube (the beads were washed twice with sorting buffer before use and then resuspended in the same volume of sorting buffer as the original volume), mix thoroughly, and incubate at 4°C for 5 min.

[0129] 5. After incubation, transfer the cells to a flow cytometry tube, add separation buffer to 2.5 mL, and mix thoroughly by pipetting up and down.

[0130] 6. Place the cell separation tube on the magnetic stand and let it stand for 2 minutes.

[0131] 7. Gently pour the cell suspension into a sterile centrifuge tube. This cell suspension contains the purified mouse neutrophils.

[0132] The STEMCELL sorting product uses the following steps to complete the sorting process in 20 minutes:

[0133] 1. Add 100 μL of mouse bone marrow cell suspension (1×10 7 cells) were added to the bottom of a 1.5 mL centrifuge tube, and then 5 uL of Enrichment cocktail was added, mixed, and incubated at 4°C for 5 min.

[0134] 2. After incubation, wash the cells once, add sorting buffer to 1.5 mL, and centrifuge at 500 g for 3 min (sorting buffer is PBS containing 2 mM EDTA and 2% fetal bovine serum (FBS)).

[0135] 3. After centrifugation, discard the supernatant and resuspend the cells in 100 μL of sorting buffer.

[0136] 4. Then add Biotin Selection Cocktail to the centrifuge tube, mix well, and incubate at 4°C for 5 min.

[0137] 5. Add 15 μL of thoroughly mixed Magnetic Particles into the centrifuge tube, mix thoroughly, and incubate at 4°C for 5 minutes.

[0138] 6. After incubation, transfer the cells to a flow cytometry tube, add separation buffer to 2.5 mL, and mix thoroughly by pipetting up and down.

[0139] 7. Place the cell separation tube on the magnetic stand and let it stand for 2 minutes.

[0140] 8. Gently pour the cell suspension containing the purified mouse neutrophils into a sterile flow cytometry tube.

[0141] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composition of a biotin-labeled antibody, characterized in that: The antibodies include: CD4, CD5, bCD19, CD16 / 32, CD117 and bAB20.

2. The composition according to claim 1, characterized in that The concentration of the biotin-labeled CD4 is 0.1 mg / mL to 1 mg / mL; the concentration of the biotin-labeled CD5 is 0.1 mg / mL to 1 mg / mL; the concentration of the biotin-labeled bCD19 is 0.1 mg / mL to 1 mg / mL; the concentration of the biotin-labeled CD16 / 32 is 0.1 mg / mL to 1 mg / mL; the concentration of the biotin-labeled CD117 is 0.1 mg / mL to 1 mg / mL; and the concentration of the biotin-labeled bAB20 is 0.1 mg / mL to 1 mg / mL.

3. A reagent, characterized in that The invention comprises the composition according to claim 1 or 2 and auxiliary materials.

4. A kit, characterized in that It comprises at least one of the composition according to claim 1 or 2 and / or the reagent according to claim 3, a detergent and / or a sorting buffer.

5. The kit according to claim 4, characterized in that The sorting buffer is selected from at least one of the following: PBS containing EDTA and fetal bovine serum; and / or PBS containing EDTA and bovine serum albumin.

6. Application of any one of the following I) to III) in neutrophil sorting: 1), a composition according to claim 1 or 2; II), the reagent according to claim 3; III), the kit according to claim 4 or 5.

7. A method for separating neutrophils, characterized in that: The method comprises sorting neutrophils in a sample using at least one of the following i) to iii): i), the composition according to claim 1 or 2; ii), the reagent according to claim 3; iii), the kit according to claim 4 or 5.

8. The method according to claim 7, characterized in that The steps include: Step 1, mixing the sample with the composition according to claim 1 or 2 and then performing a first incubation; Step 2, the product after the first incubation is subjected to a second incubation with streptavidin-labeled magnetic beads; Step 3: performing magnetic separation on the product after the second incubation to obtain the neutrophils.

9. The method according to claim 8, characterized in that The concentration of cells in the sample was 1×10 8 / mL; The first incubation condition is 2°C to 6°C for 5min to 15min; The second incubation condition is 2°C to 6°C for 5min to 15min; The magnetic separation time is 4 to 6 minutes; After the first incubation in step 1 and before step 2, the steps of washing and centrifugation are also included.

10. The method according to any one of claims 7 to 8, characterized in that: The sample includes mouse bone marrow cells, spleen cells and / or peripheral blood.

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