CN113969261B - Method for rapidly determining chemotaxis of neutrophils by three-step method - Google Patents
Method for rapidly determining chemotaxis of neutrophils by three-step method Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
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- G—PHYSICS
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- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5047—Cells of the immune system
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention relates to a three-step method for rapidly determining chemotaxis of neutrophils, which comprises the following steps: s1, adding a dextran solution with the concentration of 2.8% -3% into a whole blood sample to settle red blood cells, wherein the volume of the added dextran solution is 1.1-1.3 times of the volume of the whole blood sample, and centrifuging the supernatant; s2, centrifuging, discarding supernatant, adding sterilized water for injection to treat red blood cells, and centrifuging to obtain neutrophils; s3, counting the centromere cells, and taking the cell suspension to a chemotactic model for chemotaxis. The three-step method for rapidly determining the chemotaxis of the neutrophils can effectively maintain the activity of the neutrophils, and has the advantages of short time and low cost.
Description
Technical Field
The invention relates to a method for rapidly determining chemotaxis of neutrophils, and belongs to the technical field of biology.
Background
Neutrophils are important congenital immune cells of human body for resisting pathogen invasion, are produced by bone marrow hematopoietic stem cells, are differentiated and developed in bone marrow, enter blood or tissues, are white blood cells with the largest quantity in peripheral blood of an organism, and account for 40% -75% of total white blood cells under normal physiological state. In the blood smear of Rayleigh staining, the cytoplasm is colorless or light red, the nucleus is rod-shaped or 2-5-minute leaf-shaped, and the leaves are connected by filaments, so that the neutrophil is also commonly called polymorphonuclear leucocyte (polymorphonuclear leukocyte, PMN) and has the functions of chemotaxis, phagocytosis and sterilization. When the human body is affected by pathogenic bacteria, neutrophils can be chemotactic to the infected part, bacteria are swallowed into the cells, and the bacteria are killed through various ways, so that the neutrophil is a first defense line for resisting bacterial infection of the human body, and plays a very important role in the non-specific immune system of the human body. Chemotaxis (also known as chemotropism) is a type of chemotaxis that refers to the trending of organisms to the stimulation of chemical substances in the external environment, and the movement of body cells, bacteria and other single-cell, multicellular organisms tend to depend on certain chemical substances in the environment.
In recent years, a great deal of domestic and foreign researches show that the trouble of various diseases is closely related to neutrophils at present: abnormal chemotaxis of neutrophils in infected patients, which makes neutrophils unable to reach the infected site to clear pathogenic microorganisms, while excessive neutrophils recruit to non-inflammatory sites to cause organ damage; neutrophil release dysfunction of patients with tumor, alkaline phosphatase released by neutrophil has anti-tumor effect, and when it is released excessively, it can cause tissue injury, destroy pericancerous tissue, and provide favorable condition for tumor diffusion; the neutrophil death mode of the diabetic patient is abnormal, the neutrophil extracellular trap net (Neutrophil extracellular traps, NETs) is excessively formed on the wound surface of the diabetic patient, and the proinflammatory cell death mode leads to difficult healing of the chronic wound surface of the diabetic patient and the like. Therefore, the detection of neutrophil function provides new insight for pathophysiology of various diseases, provides new direction for treatment of diseases, and has very important clinical significance.
Phagocytosis of pathogens by neutrophils generally involves several steps of chemotaxis, opsonization, phagocytosis and sterilization. Under the action of chemotactic factors, the neutrophils move to the periphery of bacteria in a directional way, the bacteria subjected to opsonin action are easy to adhere to the neutrophils, so that neutrophil membranes are sunk, the bacteria are swallowed through pinocytosis to form phagocytic packets, and the phagocytic packets are fused with lysosomes in cells to form lysosomes, so that the bacteria are killed. Therefore, the chemotactic ability of neutrophils is the core of the sterilization effect, and the decrease of the chemotactic ability of neutrophils can lead to the failure of neutrophils to reach the infection position, so that the organism is easy to infect.
The procedure for extracting neutrophils and performing chemotaxis experiments using the conventional five-step method is as follows: 1. centrifuging after sedimentation of erythrocytes; 2. polysucrose density gradient centrifugation; 3. discarding the peripheral blood mononuclear cell layer; 4. lysing erythrocytes (two lyses were required); 5. cell count, chemotaxis assay. The conventional five-step method is used for extracting the neutrophils and detecting the neutrophils, at least 4 hours is needed, and the cost is high.
The procedure for the conventional five-step extraction of neutrophils and cell count agarose cell chemotaxis assay can be found in the following references: the effect of endotoxin/lipopolysaccharide on early apoptosis of human neutrophils by PIM3 is described in journal of Chinese burn, volume 34, 11 th, pages 809-814 of 2018, 11 th month; the mechanism study of the delay of the apoptosis of peripheral blood neutrophils of patients with severe burns is called "Chinese critical illness emergency medicine, 10 th month 31, 10 th phase 1208-1211 in 2019; the university of Jiangsu university journal (medical edition) 2018, 11 th month, 28 th volume, 6 th phase 467-472 pages; adenosine effectively restores endotoxin-induced inhibition of human neutrophil chemotaxis via A1 receptor-p38 pathway (adenosine is effective in restoring chemotaxis of endotoxin-induced inhibition of the p38 pathway of the A1 receptor by human neutrophils) Inflammation ResearchOfficial Journal of: the InternationalAssociation of Inflammation Societies + The European Histamine ResearchSociety ISSN 1023-3830 Volume 66 Number 4 April 2017 (journal of the society of inflammation research: international society of inflammation+European Histamine research society ISSN 1023-3830, 2017, month, volume 66, pages 4, 353-364).
Disclosure of Invention
The invention aims to provide a three-step method for rapidly determining chemotaxis of neutrophils, which can effectively maintain the activity of the neutrophils, and has the advantages of short time and low cost.
In order to solve the technical problems, the invention provides a three-step method for rapidly determining neutrophil chemotaxis, which comprises the following steps:
s1, adding a dextran solution with the concentration of 2.8% -3% into a whole blood sample to settle red blood cells, wherein the volume of the added dextran solution is 1.1-1.3 times of the volume of the whole blood sample, and centrifuging the supernatant;
s2, centrifuging, discarding supernatant, adding sterilized water for injection to treat red blood cells, and centrifuging to obtain neutrophils;
s3, counting the centromere cells, and taking the cell suspension to a chemotactic model for chemotaxis.
The added volume of the dextran solution with the concentration of 3 percent is 1.2 to 1.3 times of the volume of the whole blood sample.
In the step S1, 1.2mL of dextran solution is added to 1mL of whole blood sample, the mixture is inverted and mixed uniformly, and the mixture is left at room temperature, and then the supernatant is transferred to a centrifuge tube for centrifugation.
The standing time at room temperature is 15 min-20 min.
The centrifugation time of the step S1 is 7-10 min, the centrifugal force is 400-450 g, and the centrifugation temperature is 20-25 ℃.
And step S2, adding sterilized water for injection to treat erythrocytes, then blowing and sucking, sequentially adding a balanced salt solution with the concentration of 2 times and a balanced salt solution with the concentration of 1 time, reversing and uniformly mixing, and centrifuging to obtain the neutrophils.
The dosage of the sterilizing water for injection, the balanced salt solution with the concentration of 2 times and the balanced salt solution with the concentration of 1 time is 2.5 mL-3.5 mL.
The blowing and sucking time is 25-30 s.
The centrifugation time of the step S2 is 7-10 min, the centrifugal force is 400-450 g, and the centrifugation temperature is 20-25 ℃.
The step S3 is to count the granulocytes first and then adjust the cell concentration to 1X 10 6 ～1×10 7 mu.L to 10. Mu.L of the cell suspension was taken per mL and chemotactic in an agarose cell chemotactic model.
The invention has the positive effects that: the three-step method for rapidly determining the chemotaxis of the neutrophils of the invention accelerates sedimentation of the erythrocytes by adding more glucan solution, saves time cost, only takes 50 minutes for extracting the cells, saves about 70 minutes compared with the conventional five-step method, better maintains the activity of the neutrophils, can simplify experimental steps, does not remove peripheral blood mononuclear cell layers in the leukocytes, better accords with chemotaxis environment of the neutrophils in human body, saves about 75 percent of cost, and has no difference with the chemotaxis experimental results obtained by the conventional five-step method. In addition, the method has small demand for blood samples, and can reduce the burden of human bodies.
Drawings
FIG. 1 is a graph of the chemotactic distance of neutrophils 2h using the three-step rapid chemotactic method of example 1 of the present invention;
FIG. 2 is a graph of the 2h chemotactic distance of neutrophils according to the five-step chemotactic method of comparative example 1 of the present invention;
fig. 3 is a graph comparing chemotactic distances of fig. 1 and 2.
Detailed Description
The present invention is described in detail below by way of examples, which are necessary to be pointed out herein for further illustration of the invention and are not to be construed as limiting the scope of the invention, since numerous insubstantial modifications and adaptations of the invention will be to those skilled in the art in light of the foregoing disclosure. In the examples which follow, reagents used were all analytically pure and were all available from commercial sources unless specifically indicated. The experimental methods for which specific conditions are not specified are generally performed according to conventional conditions or according to conditions recommended by the manufacturer. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention.
Example 1
The three-step method for rapidly determining neutrophil chemotaxis of the embodiment comprises the following specific steps:
s1, centrifuging after sedimentation of erythrocytes.
Whole blood samples were collected with EDTA (ethylenediamine tetraacetic acid) anticoagulation tubes, 1mL per tube, then 1.2mL of DEX (dextran solution) at a concentration of 3% was added, blood sedimentation was accelerated, mixing was slowly reversed, standing at room temperature for 15min, and the supernatant was transferred to a 15mL centrifuge tube for centrifugation at a centrifugal force of 400g for 10min at a centrifugal temperature of 20 ℃.
S2, lysing the red blood cells.
Centrifuging, discarding supernatant, treating erythrocytes in cell mass with 3mL of sterilized water for injection, gently blowing and sucking for 30s, sequentially adding 3mL of 2-time concentration HBSS (Hank's balanced salt solution without calcium and magnesium ions) and 3mL of 1-time concentration HBSS, mixing uniformly, centrifuging to obtain neutrophils, centrifuging for 400g for 7min at 20 ℃.
S3, cell counting and chemotaxis experiments.
For neutrophilsCount, then adjust cell concentration to 1 x 10 7 mu.L of the cell suspension was taken at a concentration of one liter per mL and chemotaxis was performed in an agarose cell chemotaxis model.
In this example, the extraction time of the cells was about 50 minutes, and the chemotaxis test was about 2 hours.
In the step S1, most of red blood cells can be settled and removed, in the step S2, residual red blood cells are lysed, the total removal of the red blood cells is ensured, in the step S3, the number of the granulocytes is counted, the cell concentration is adjusted through dilution or concentration, and a cell suspension is taken, and then chemotactic experiments are carried out.
Example 2
The three-step method for rapidly determining neutrophil chemotaxis of the embodiment comprises the following specific steps:
s1, centrifuging after sedimentation of erythrocytes.
Whole blood samples were collected with EDTA (ethylenediamine tetraacetic acid) anticoagulation tubes, 1mL per tube, then 1.3mL of DEX (dextran solution) at a concentration of 2.8% was added, blood sedimentation was accelerated, mixing was slowly reversed, standing at room temperature for 15min, and the supernatant was transferred to a 15mL centrifuge tube for centrifugation at a centrifugal force of 400g for 10min at a centrifugal temperature of 20 ℃.
S2, lysing the red blood cells.
Centrifuging, discarding supernatant, treating erythrocytes in cell mass with 3mL of sterilized water for injection, gently blowing and sucking for 30s, sequentially adding 3mL of 2-time concentration HBSS (Hank's balanced salt solution without calcium and magnesium ions) and 3mL of 1-time concentration HBSS, mixing uniformly, centrifuging to obtain neutrophils, centrifuging for 400g for 7min at 20 ℃.
S3, cell counting and chemotaxis experiments.
Neutrophils were counted and then cell concentration was adjusted to 1 x 10 7 mu.L of the cell suspension was taken at a concentration of one liter per mL and chemotaxis was performed in an agarose cell chemotaxis model.
Example 3
The three-step method for rapidly determining neutrophil chemotaxis of the embodiment comprises the following specific steps:
s1, centrifuging after sedimentation of erythrocytes.
Whole blood samples were collected with EDTA (ethylenediamine tetraacetic acid) anticoagulation tubes, 1mL per tube, then 1.1mL of DEX (dextran solution) at a concentration of 2.9% was added, blood sedimentation was accelerated, mixing was slowly reversed, standing at room temperature for 15min, and the supernatant was transferred to a 15mL centrifuge tube for centrifugation at a centrifugal force of 400g for 10min at a centrifugal temperature of 20 ℃.
S2, lysing the red blood cells.
Centrifuging, discarding supernatant, treating erythrocytes in cell mass with 3mL of sterilized water for injection, gently blowing and sucking for 30s, sequentially adding 3mL of 2-time concentration HBSS (Hank's balanced salt solution without calcium and magnesium ions) and 3mL of 1-time concentration HBSS, mixing uniformly, centrifuging to obtain neutrophils, centrifuging for 400g for 7min at 20 ℃.
S3, cell counting and chemotaxis experiments.
Neutrophils were counted and then cell concentration was adjusted to 1 x 10 7 mu.L of the cell suspension was taken at a concentration of one liter per mL and chemotaxis was performed in an agarose cell chemotaxis model.
Comparative example 1
The five-step method of the comparative example is a method for extracting neutrophils and chemotactic, comprising the following specific steps:
s1, centrifuging after sedimentation of erythrocytes.
Whole blood samples were collected with EDTA (ethylenediamine tetraacetic acid) anticoagulation tubes, 2mL of each tube, then 2mL of DEX (dextran solution) at a concentration of 3% was added, mixed upside down, and left standing at room temperature for 20min, and the supernatant was transferred to a 15mL centrifuge tube for centrifugation at a centrifugal force of 400g for 10min at a centrifugal temperature of 20 ℃.
S2, ficoll density gradient centrifugation.
The bottom cell pellet was resuspended with 3mL of HBSS at a concentration doubling, and after uniform blowing, 3mL of Ficoll (polysucrose) was slowly added from the bottom of the centrifuge tube (protected from light), density gradient centrifuged, centrifugal force 400g, centrifugation time 35min, centrifugation temperature 20 ℃.
S3. The PBMC (Peripheral Blood Mononuclear Cell, peripheral blood mononuclear cells) layer was discarded.
After centrifugation, the liquid level is divided into two layers, wherein the middle layer is a PBMC layer containing a large number of monocytes, lymphocytes and the like, and the bottom is red blood cells and mature neutrophils. The PBMC layer was carefully aspirated and the other supernatant was aspirated.
S4, lysing the erythrocytes (twice).
Treating erythrocytes in the cell mass by adopting 3mL of sterilized water for injection, gently blowing and sucking for 30s, sequentially adding 3mL of HBSS with 2 times concentration and 3mL of HBSS with 1 times concentration, reversing and uniformly mixing, centrifuging to obtain neutrophils, centrifuging for 400g, centrifuging for 7min, and centrifuging at 20 ℃.
S5, cell counting and chemotaxis experiments.
Neutrophils were counted and then cell concentration was adjusted to 1 x 10 7 mu.L of the cell suspension was taken at a concentration of one liter per mL and chemotaxis was performed in an agarose cell chemotaxis model.
The comparative example 1 took about 120min for cell extraction and about 2h for chemotaxis experiments.
FIG. 1 is a graph showing the chemotactic distance of neutrophils 2h according to the three-step rapid chemotactic method of example 1, wherein the distance a is 1865.6 μm in length, and FIG. 2 is a graph showing the chemotactic distance of neutrophils 2h according to the five-step chemotactic method, wherein the distance b is 1850.2. Mu.m in length. For example, as shown in fig. 3, the sample number n=8, and the unpaired t test is adopted, so that p=0.8548 >0.05 is obtained, that is, the chemotactic distances of the neutrophils obtained by the two experimental schemes are not different.
The HBSS balanced salt solution is a Gbico brand Hank's balanced salt solution without calcium and magnesium ions. The dextran solution with the concentration of 3% is prepared by mixing dextran with Hank's balanced salt solution without calcium and magnesium ions. Ficoll polysucrose solution is of the GE brand.
The cell count is carried out by a full-automatic cell counter, an agarose cell chemotaxis model is constructed by a conventional agarose chemotaxis experiment method, an agarose block is prepared by 1640 medium containing 10% of the fetal bovine serum FBS, and the fetal bovine serum FBS and the 1640 medium are both of Gbico brands.
Claims (8)
1. A method for rapidly determining neutrophil chemotaxis by a three-step method for non-diagnostic purposes, comprising the steps of:
s1, adding a dextran solution with the concentration of 2.8% -3% into a whole blood sample to settle red blood cells, wherein the volume of the added dextran solution is 1.1-1.3 times of the volume of the whole blood sample, standing at room temperature, and centrifuging supernatant, wherein the standing time at room temperature is 15-20 min;
s2, centrifuging, discarding supernatant, adding sterilized water for injection to treat erythrocytes, blowing and sucking, sequentially adding balanced salt solution with the concentration of 2 times and balanced salt solution with the concentration of 1 time, reversing and uniformly mixing, and centrifuging to obtain neutrophils;
s3, counting the centromere cells, and taking the cell suspension to a chemotactic model for chemotaxis.
2. The method for rapid determination of neutrophil chemotaxis by a three-step method for non-diagnostic purposes according to claim 1, wherein: the adding volume of the dextran solution with the concentration of 3% is 1.2-1.3 times of the whole blood sample volume.
3. The method for rapid determination of neutrophil chemotaxis by a three-step method for non-diagnostic purposes according to claim 2, characterized in that: step S1 is to add 1.2mL of dextran solution into 1mL of whole blood sample, mix the mixture upside down, stand the mixture at room temperature, and then transfer the supernatant into a centrifuge tube for centrifugation.
4. A method for the rapid determination of neutrophil chemotaxis in a three-step method for non-diagnostic purposes according to claim 3, characterized in that: the centrifugation time of the step S1 is 7-10 min, the centrifugal force is 400-450 g, and the centrifugation temperature is 20-25 ℃.
5. The method for rapid determination of neutrophil chemotaxis by a three-step method for non-diagnostic purposes according to claim 1, wherein: the dosage of the sterilizing water for injection, the balanced salt solution with the concentration of 2 times and the balanced salt solution with the concentration of 1 time is 2.5-mL-3.5 mL.
6. The method for rapid determination of neutrophil chemotaxis by a three-step method for non-diagnostic purposes according to claim 1, wherein the time of blowing-up is 25s to 30s.
7. The method for rapid determination of neutrophil chemotaxis by three-step method for non-diagnostic purposes according to claim 1, wherein the centrifugation time of step S2 is 7 min-10 min, the centrifugation force is 400 g-450 g, and the centrifugation temperature is 20 ℃ to 25 ℃.
8. The method for rapid determination of neutrophil chemotaxis by a three-step method for non-diagnostic purposes according to claim 1, wherein: the step S3 is to count the granulocytes first and then adjust the cell concentration to 1X 10 6 ～1×10 7 mu.L to 10. Mu.L of the cell suspension was taken per mL and chemotactic in an agarose cell chemotactic model.
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| CN1809374A (en) * | 2003-06-20 | 2006-07-26 | 诺瓦提斯公司 | Proton-sensing G-protein coupled receptors and DNA sequences thereof |
| CN101918594A (en) * | 2007-11-30 | 2010-12-15 | 俄亥俄州立大学研究基金会 | Micro-RNA expression profiling and targeting in peripheral blood in lung cancer |
| CN102586187A (en) * | 2012-02-23 | 2012-07-18 | 深圳市中美康士生物科技有限公司 | In vitro preservation method and culture medium for neutrophils |
| CN104519879A (en) * | 2012-06-07 | 2015-04-15 | 洛杉矶儿童医院 | Methods for treating neutropenia using retinoid agonists |
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| CN1809374A (en) * | 2003-06-20 | 2006-07-26 | 诺瓦提斯公司 | Proton-sensing G-protein coupled receptors and DNA sequences thereof |
| CN101918594A (en) * | 2007-11-30 | 2010-12-15 | 俄亥俄州立大学研究基金会 | Micro-RNA expression profiling and targeting in peripheral blood in lung cancer |
| CN102586187A (en) * | 2012-02-23 | 2012-07-18 | 深圳市中美康士生物科技有限公司 | In vitro preservation method and culture medium for neutrophils |
| CN104519879A (en) * | 2012-06-07 | 2015-04-15 | 洛杉矶儿童医院 | Methods for treating neutropenia using retinoid agonists |
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