Isolated culture method for primary neutrophil of mouse aorta

Abstract

The invention discloses a mouse aorta primary neutrophile granulocyte isolated culture method, and relates to the technical field of neutrophile granulocyte isolated culture, and the method specifically comprises the following steps: 1, anesthetizing a mouse, and placing the mouse in 700-750mL / L ethanol to soak and disinfect for 5-8min; 2, under the sterile environment condition, opening the thoracic cavity of the mouse, separating the aorta, placing the mouse in a precooled PBS solution, cleaning blood, and removing fat and connective tissue on the periphery of the blood vessel; and step 3, cutting the cleaned aorta into blocks of 1-2mm < 3 >, then putting the blocks into a centrifugal tube, adding a mixed digestive juice, digesting in a water bath at 37 DEG C for 30-60 minutes, gently oscillating every 10 minutes during the digesting, then adding a DMEM / F12 culture solution to terminate the digesting, and then sequentially centrifuging and filtering to obtain the cell suspension. According to the mouse aorta primary neutrophil isolated culture method, aorta tissue is flushed with PBS containing double antibodies, bacteria, fungi and other microorganisms possibly contaminated in the material taking process are effectively killed, and the contamination probability in the cell culture process is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of neutrophil separation and culture, and in particular to a method for separating and culturing primary neutrophils of mouse aorta. Background Art

[0002] Mouse aortic primary neutrophils refer to neutrophils isolated from the mouse aorta. These cells have multiple biological functions in the body, including phagocytosis of pathogens, participation in immune responses, etc.; by isolating and culturing neutrophils, it is helpful to deeply analyze the molecular mechanisms and signal pathways of the specific functions of neutrophils in the physiological environment of the aorta, such as chemotaxis, phagocytosis, and immune regulation, and to explore their interaction with aortic endothelial cells, smooth muscle cells, etc., such as how neutrophils affect the functional state of endothelial cells by secreting cytokines, which provides a basis for the early diagnosis of aortic-related diseases, the discovery of therapeutic targets, and the optimization of treatment plans, and is of great significance for the biological research of neutrophils and their clinical application; however, in the prior art, there is little research on the isolation and culture technology of aortic primary neutrophils. To this end, the present invention proposes a method for isolating and culturing mouse aortic primary neutrophils to solve the above-mentioned problems. Summary of the invention

[0003] 1. Technical issues to be resolved

[0004] In view of the deficiencies in the prior art, the present invention provides a method for isolating and culturing primary neutrophils from mouse aorta, which solves the problems mentioned in the above background technology.

[0005] (II) Technical solution

[0006] To achieve the above objectives, the present invention is implemented by the following technical scheme: a method for isolating and culturing primary neutrophils from mouse aorta, specifically comprising the following steps:

[0007] Step 1: After anesthetizing the mouse, soak it in 700-750mL / L ethanol for 5-8 minutes;

[0008] Step 2: Under sterile conditions, open the mouse chest cavity, isolate the aorta, wash the blood in a pre-cooled PBS solution, and remove the fat and connective tissue around the blood vessels;

[0009] Step 3: Cut the cleaned aorta into 1-2 mm pieces 3 The cells were then placed in a centrifuge tube and mixed with digestion solution. The cells were digested in a 37°C water bath for 30-60 min, with gentle shaking every 10 min. DMEM / F12 culture medium was then added to terminate the digestion. The cells were then centrifuged and filtered to obtain a cell suspension.

[0010] Step 4: Use physiological saline to dilute the Percoll separation solution into two concentrations of 70% and 40%, first add 70% Percoll solution to the centrifuge tube, then layer 40% Percoll solution on the upper layer, layer the cell suspension on the upper layer of the Percoll gradient solution, then centrifuge, and absorb the neutrophils at the interface of 40% and 70% Percoll solution;

[0011] Step 5: Place the collected neutrophils in a 45-50 mL culture bottle covered with rat tail glue, add DMEM / F12 culture medium, and then place it in an incubator for static culture for 6-8 days, then discard the aorta and replace the medium;

[0012] Step 6. After 12-14 days of culture, aspirate the supernatant and add DMEM / F12 culture medium at a ratio of 1 / 4-1 / 3 according to the amount of cells, and then change the medium every other day.

[0013] Preferably, in step 2, the PBS solution contains 1%-2% of blue chain bispecific antibody.

[0014] Preferably, the DMEM / F12 culture medium contains 20%-25% newborn calf serum, and the amount of culture medium added is only enough to cover the aorta.

[0015] Preferably, in step three, the mixed digestion solution includes 0.1% collagenase II and 0.05% trypsin.

[0016] Preferably, in step three, centrifugation is performed at a speed of 900-1000 r / min for 5-10 min, and filtration is performed using a 60-70 μm cell sieve.

[0017] Preferably, in step 4, centrifugation is performed at a speed of 1800-2000 r / min for 20-30 min.

[0018] Preferably, in step five, the temperature in the incubator is 37° C., the content of C02 is 5%-7%, and the humidity in the incubator is saturated.

[0019] Preferably, in step 5, the preparation method of rat tail glue is:

[0020] (1) After killing the mice by cervical dislocation, cut off the tails under sterile conditions;

[0021] (2) First, disinfect the mouse tail by soaking it in 75% alcohol for 5-8 minutes, then rinse the mouse tail with sterile PBS, and then fix the mouse tail with 95% ethanol for 10-15 minutes;

[0022] (3) Remove the tail muscle and connective tissue, disinfect them with 75% ethanol for 25-30 min, and then rinse them repeatedly with sterile triple-distilled water until no ethanol residue remains;

[0023] (4) Cut the rat tail into small pieces, place them in 100 mL of 0.1% acetic acid solution, stir and extract at 4°C for 44-48 hours, then aspirate the upper layer of liquid, centrifuge, take the supernatant, and filter it through a 0.22-0.25 μm filter membrane to obtain the rat tail glue solution.

[0024] Preferably, in (4), the centrifugal speed is 2500-3000 r / min, and the centrifugal time is 30-35 min.

[0025] (III) Beneficial effects

[0026] The present invention provides a method for isolating and culturing primary neutrophils from mouse aorta. Compared with the prior art, the method has the following beneficial effects: the method for isolating and culturing primary neutrophils from mouse aorta can effectively kill bacteria, fungi and other microorganisms that may be contaminated during the sampling process by flushing the aortic tissue with PBS containing double antibodies, reduce the probability of contamination during the cell culture process, and ensure that the cells grow in a relatively sterile environment; use DMEM / F12 culture medium containing newborn calf serum to terminate digestion; the protein in the serum can neutralize the digestive enzyme, quickly stop the digestion reaction, prevent excessive digestion from damaging the cells, and help maintain the normal morphology and physiological function of the cells; use 40% and 70% concentrations of Percoll solution for density gradient centrifugation, and neutrophils can be effectively separated from other cell components according to the density difference of the cells, so that neutrophils with higher purity can be obtained. cells, reducing interference from other cell types, which is conducive to specialized research on neutrophils; by culturing cells in culture bottles covered with rat tail glue, rat tail glue provides cells with a natural extracellular matrix environment, which is conducive to the adhesion and growth of neutrophils, can better simulate the in vivo environment, and enable cells to maintain their original biological characteristics; using DMEM / F12 culture medium provides cells with rich and balanced nutrients to meet the needs of neutrophil growth, metabolism and proliferation, and is conducive to long-term culture of cells in vitro and maintaining their functional activity; by changing the medium every other day, waste and toxins produced by cell metabolism can be removed in time, fresh nutrients can be added, and the stability of the cell growth environment can be maintained, which is conducive to the long-term survival and stable growth of cells and convenient for long-term experimental observation and research. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0028] Example 1

[0029] A method for isolating and culturing primary neutrophils from mouse aorta, specifically comprising the following steps:

[0030] Step 1: After anesthetizing the mice, soak them in 700 mL / L ethanol for 5 min;

[0031] Step 2: Under sterile conditions, open the mouse chest cavity, isolate the aorta, wash the blood in a pre-cooled PBS solution containing 1% cyanine chain double antibody, and remove the fat and connective tissue around the blood vessels;

[0032] Step 3: Cut the cleaned aorta into 1mm pieces 3 The cells were then placed in a centrifuge tube and digested in a mixed digestion solution of 0.1% collagenase II and 0.05% trypsin at 37°C for 30 minutes in a water bath, with gentle shaking every 10 minutes. The cells were then added with DMEM / F12 culture medium containing 20% ​​newborn calf serum to terminate the digestion. The cells were then centrifuged at 900 r / min for 5 minutes and filtered through a 60 μm cell sieve to obtain a cell suspension.

[0033] Step 4: Use physiological saline to dilute the Percoll separation solution into two concentrations of 70% and 40%, first add 70% Percoll solution to the centrifuge tube, then layer 40% Percoll solution on the upper layer, layer the cell suspension on the upper layer of the Percoll gradient solution, and then centrifuge at 1800r / min for 20min, and absorb the neutrophils at the interface of 40% and 70% Percoll solution;

[0034] Step 5: Place the collected neutrophils in a 45 mL culture bottle covered with rat tail glue, add DMEM / F12 culture medium containing 20% ​​newborn calf serum, and then place it in an incubator at 37°C, 5% CO2 content, and saturated humidity for 6 days, then discard the aorta and replace the medium;

[0035] The preparation method of rat tail glue is as follows: after killing mice by cervical dislocation, cut off the rat tail under sterile conditions; first, disinfect the rat tail by soaking it in 75% alcohol for 5 minutes, then rinse the rat tail with sterile PBS, and then fix the rat tail with 95% ethanol for 10 minutes; remove the muscle and connective tissue of the rat tail, disinfect it with 75% ethanol for 25 minutes, and then repeatedly rinse it with sterile triple-distilled water until there is no ethanol residue; cut the rat tail into small pieces, place it in 100mL of 0.1% acetic acid solution, stir and extract it at a temperature of 4°C for 44 hours, then absorb the upper liquid, centrifuge it at a speed of 2500r / min for 30 minutes, and then take the supernatant, filter it through a 0.22μm filter membrane to obtain a rat tail glue solution;

[0036] Step 6: After 12 days of culture, aspirate the supernatant and add DMEM / F12 culture medium containing 20% ​​newborn calf serum. Culture at 1 / 4 of the volume of cells, and then change the medium every other day.

[0037] Example 2

[0038] A method for isolating and culturing primary neutrophils from mouse aorta, specifically comprising the following steps:

[0039] Step 1: After anesthetizing the mice, soak them in 720 mL / L ethanol for 6 minutes for disinfection;

[0040] Step 2: Under sterile conditions, the mouse chest cavity was opened, the aorta was isolated, and the aorta was placed in a pre-cooled PBS solution containing 1.5% cyanine chain double antibody to wash the blood, and the fat and connective tissue around the blood vessels were removed;

[0041] Step 3: Cut the cleaned aorta into 1.5 mm pieces 3 The cells were then placed in a centrifuge tube and digested in a mixed digestion solution of 0.1% collagenase II and 0.05% trypsin at 37°C for 40 minutes with gentle shaking every 10 minutes. The cells were then added with DMEM / F12 culture medium containing 22% newborn calf serum to terminate the digestion. The cells were then centrifuged at 950 r / min for 8 minutes and filtered through a 65 μm cell sieve to obtain a cell suspension.

[0042] Step 4: Use physiological saline to dilute the Percoll separation solution into two concentrations of 70% and 40%, first add 70% Percoll solution to the centrifuge tube, then layer 40% Percoll solution on the upper layer, layer the cell suspension on the upper layer of the Percoll gradient solution, and then centrifuge at 1900r / min for 25min, and absorb the neutrophils at the interface of 40% and 70% Percoll solution;

[0043] Step 5: Place the collected neutrophils in a 48 mL culture bottle covered with rat tail glue, add DMEM / F12 culture medium containing 23% newborn calf serum, and then place it in an incubator at 37°C, 6% CO2 content, and saturated humidity for 7 days, then discard the aorta and replace the medium;

[0044] The preparation method of rat tail glue is as follows: after killing the mouse by cervical dislocation, the rat tail is cut off under sterile conditions; the rat tail is first disinfected by soaking in 75% alcohol for 6 minutes, then rinsed with sterile PBS, and then fixed with 95% ethanol for 12 minutes; the muscle and connective tissue of the rat tail are removed, and the rat tail is disinfected with 75% ethanol for 28 minutes, and then repeatedly rinsed with sterile triple-distilled water until no ethanol residue is left; the rat tail is cut into small pieces, placed in 100 mL of 0.1% acetic acid solution, stirred and extracted for 46 hours at a temperature of 4°C, and then the upper liquid is absorbed, centrifuged at a speed of 2800r / min for 32 minutes, and then the supernatant is taken and filtered through a 0.24μm filter membrane to obtain a rat tail glue solution;

[0045] Step 6: After 13 days of culture, aspirate the supernatant and add DMEM / F12 culture medium containing 24% newborn calf serum. Culture at 1 / 3 of the volume of cells, and then change the medium every other day.

[0046] Example 3

[0047] A method for isolating and culturing primary neutrophils from mouse aorta, specifically comprising the following steps:

[0048] Step 1: After anesthetizing the mice, soak them in 750 mL / L ethanol for 8 minutes for disinfection;

[0049] Step 2: Under sterile conditions, open the mouse chest cavity, isolate the aorta, wash the blood in a pre-cooled PBS solution containing 2% cyanine chain double antibody, and remove the fat and connective tissue around the blood vessels;

[0050] Step 3: Cut the cleaned aorta into 2 mm pieces 3 The cells were then placed in a centrifuge tube and digested in a mixed digestion solution of 0.1% collagenase II and 0.05% trypsin at 37°C for 60 minutes with gentle shaking every 10 minutes. The cells were then added with DMEM / F12 culture medium containing 25% newborn calf serum to terminate the digestion. The cells were then centrifuged at 1000 r / min for 10 minutes and filtered through a 70 μm cell sieve to obtain a cell suspension.

[0051] Step 4: Use physiological saline to dilute the Percoll separation solution into two concentrations of 70% and 40%, first add 70% Percoll solution to the centrifuge tube, then layer 40% Percoll solution on the upper layer, layer the cell suspension on the upper layer of the Percoll gradient solution, and then centrifuge at 2000r / min for 30min, and absorb the neutrophils at the interface of 40% and 70% Percoll solution;

[0052] Step 5: Place the collected neutrophils in a 50 mL culture bottle covered with rat tail glue, add DMEM / F12 culture medium containing 25% newborn calf serum, and then place it in an incubator at 37°C, 7% CO2 content, and saturated humidity for 8 days, then discard the aorta and replace the medium;

[0053] The preparation method of rat tail glue is as follows: after killing mice by cervical dislocation, cut off the rat tail under sterile conditions; first, disinfect the rat tail by soaking it in 75% alcohol for 8 minutes, then rinse the rat tail with sterile PBS, and then fix the rat tail with 95% ethanol for 15 minutes; remove the muscle and connective tissue of the rat tail, disinfect it with 75% ethanol for 30 minutes, and then repeatedly rinse it with sterile triple-distilled water until there is no ethanol residue; cut the rat tail into small pieces, place it in 100mL of 0.1% acetic acid solution, stir and extract it at a temperature of 4°C for 48 hours, then absorb the upper layer of liquid, centrifuge it at a speed of 3000r / min for 35 minutes, and then take the supernatant, filter it through a 0.25μm filter membrane to obtain a rat tail glue solution;

[0054] Step 6: After 14 days of culture, aspirate the supernatant and add DMEM / F12 culture medium containing 25% newborn calf serum. Culture at 1 / 3 of the volume of cells, and then change the medium every other day.

[0055] Meanwhile, the contents not described in detail in this specification belong to the prior art known to those skilled in the art.

[0056] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0057] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for isolating and culturing primary neutrophils from mouse aorta, characterized in that: The specific steps include: Step 1: After anesthetizing the mouse, soak it in 700-750mL / L ethanol for 5-8 minutes; Step 2: Under sterile conditions, open the mouse chest cavity, isolate the aorta, wash the blood in a pre-cooled PBS solution, and remove the fat and connective tissue around the blood vessels; Step 3: Cut the cleaned aorta into 1-2 mm pieces 3 The cells were then placed in a centrifuge tube and mixed with digestion solution. The cells were digested in a 37°C water bath for 30-60 min, with gentle shaking every 10 min. DMEM / F12 culture medium was then added to terminate the digestion. The cells were then centrifuged and filtered to obtain a cell suspension. Step 4: Use physiological saline to dilute the Percoll separation solution into two concentrations of 70% and 40%, first add 70% Percoll solution to the centrifuge tube, then layer 40% Percoll solution on the upper layer, layer the cell suspension on the upper layer of the Percoll gradient solution, then centrifuge, and absorb the neutrophils at the interface of 40% and 70% Percoll solution; Step 5: Place the collected neutrophils in a 45-50 mL culture bottle covered with rat tail glue, add DMEM / F12 culture medium, and then place it in an incubator for static culture for 6-8 days, then discard the aorta and replace the medium; Step 6. After 12-14 days of culture, aspirate the supernatant and add DMEM / F12 culture medium at a ratio of 1 / 4-1 / 3 according to the amount of cells, and then change the medium every other day.

2. The method for isolating and culturing primary neutrophils from mouse aorta according to claim 1, characterized in that: In the step 2, the PBS solution contains 1%-2% of blue chain bispecific antibody.

3. The method for isolating and culturing primary neutrophils from mouse aorta according to claim 1, characterized in that: The DMEM / F12 culture medium contains 20%-25% newborn calf serum, and the amount of culture medium added is only enough to cover the aorta.

4. The method for isolating and culturing primary neutrophils from mouse aorta according to claim 1, characterized in that: In the step 3, the mixed digestion solution includes 0.1% collagenase II and 0.05% trypsin.

5. The method for isolating and culturing primary neutrophils from mouse aorta according to claim 1, characterized in that: In the step 3, centrifugation is performed at a speed of 900-1000 r / min for 5-10 min, and filtration is performed using a 60-70 μm cell sieve.

6. The method for isolating and culturing primary neutrophils from mouse aorta according to claim 1, characterized in that: In the step 4, centrifugation is performed at a speed of 1800-2000 r / min for 20-30 min.

7. The method for isolating and culturing primary neutrophils from mouse aorta according to claim 1, characterized in that: In step five, the temperature in the incubator is 37° C., the content of C02 is 5%-7%, and the humidity in the incubator is saturated.

8. The method for isolating and culturing primary neutrophils from mouse aorta according to claim 1, characterized in that: In the step 5, the preparation method of rat tail glue is: (1) After killing the mice by cervical dislocation, cut off the tails under sterile conditions; (2) First, disinfect the mouse tail by soaking it in 75% alcohol for 5-8 minutes, then rinse the mouse tail with sterile PBS, and then fix the mouse tail with 95% ethanol for 10-15 minutes; (3) Remove the tail muscle and connective tissue, disinfect them with 75% ethanol for 25-30 min, and then rinse them repeatedly with sterile triple-distilled water until no ethanol residue remains; (4) Cut the rat tail into small pieces, place them in 100 mL of 0.1% acetic acid solution, stir and extract at 4°C for 44-48 hours, then aspirate the upper layer of liquid, centrifuge, take the supernatant, and filter it through a 0.22-0.25 μm filter membrane to obtain the rat tail glue solution.

9. The method for isolating and culturing primary neutrophils from mouse aorta according to claim 8, characterized in that: In the above (4), the centrifugal speed is 2500-3000 r / min, and the centrifugal time is 30-35 min.