CN112210533A - Method for preparing mouse aortic artery cell single cell suspension - Google Patents
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Abstract
The invention discloses a method for preparing mouse aortic artery cell single cell suspension, which is characterized by comprising the following steps: the method specifically comprises the following steps: firstly, preparing 1ml of enzyme mixed liquor, a living healthy wild mouse, an operating table with a smooth ice surface on the whole table top and a plurality of instruments; and secondly, performing intraperitoneal injection anesthesia and death on the prepared wild mouse by using 200-500 ul chloral hydrate, immediately performing perfusion washing on blood vessels of the wild mouse by using 20ml of phosphate buffer saline solution, integrally sterilizing the dead wild mouse by using 75% alcohol by volume fraction, performing thoracoabdominal operation after sterilization is completed, and exposing the heart and the aorta, wherein the whole operation process is completed on the operation table. The method for preparing the mouse vascular tissue single cell suspension by using the shearing and mixed enzyme combined digestion method is simple, convenient and feasible, has more immune cells, and provides an effective tissue single cell suspension preparation method for the research of vascular immune cells.
Description
Technical Field
The invention relates to the technical field of quantitative cell analysis, in particular to a method for preparing mouse aortic artery cell single cell suspension.
Background
Flow cytometry is a single cell-based technique widely used in various aspects of basic research and clinical practice, such as immunophenotyping of mouse vascular cell membranes, and the preparation of single cell suspensions of mouse vascular tissues is a prerequisite for flow cytometry analysis. The existing preparation of the blood vessel tissue single cell suspension is difficult, the single cell with high yield, high activity and strong function is difficult to obtain, further experimental study is hindered, and the failure of the experiment can be caused by insufficient cell quantity, excessive cell fragments, cell adhesion and agglomeration and the like. Therefore, how to prepare a single cell suspension with better quality is a problem to be solved at present.
Disclosure of Invention
The invention aims to provide a method for preparing mouse aortic artery cell single cell suspension, which aims to solve the problems in the background technology.
In order to realize the purpose, the invention provides the following technical scheme: a method for preparing mouse aortic artery cell single cell suspension specifically comprises the following steps:
firstly, preparing 1ml of enzyme mixed liquor, a living healthy wild mouse, an operating table with a smooth ice surface on the whole table top and a plurality of instruments;
secondly, injecting 200-500 ul chloral hydrate into the abdominal cavity of the prepared wild mouse for anesthesia and death, immediately performing perfusion washing on the blood vessel of the wild mouse by using 20ml of phosphate buffer saline solution, integrally sterilizing the dead wild mouse by using alcohol with the volume fraction of 75%, performing thoracoabdominal operation after sterilization is completed, and exposing the heart and the aorta, wherein the whole operation process is completed on the operation table;
then, an aorta vessel of 11mm length was taken from the wild mouse after the thoracoabdominal operation was completed, and placed in a first centrifuge tube of 1.5mL containing 1mL of 2% FCS solution, and the aorta vessel was completely immersed in the solution, and the first centrifuge tube was placed on the operating table after the operation was completed;
then, rapidly removing fat and connective tissues around the aorta blood vessel soaked in the first centrifuge tube with the aid of a microscope, keeping the aorta blood vessel intact in the operation process, and completing the whole operation process on the operation table;
then, taking the aorta vessel after the separation of fat and connective tissue out of the first centrifuge tube, placing the aorta vessel in a second centrifuge tube of 1.5ml, adding 100ul of the enzyme-mixed digestive juice into the second centrifuge tube, completely soaking the aorta vessel in the solution, and cutting the aorta vessel into pieces by using an ophthalmic scissors, wherein the whole operation process is completed on the operation table;
then, the inner walls of the ophthalmic scissors and a second centrifuge tube are washed clean by 900ul of the enzyme mixed solution, the second centrifuge tube is tightly covered and completely placed in a 37-DEG water bath kettle for 30min, and a straw is continuously used for blowing and beating in the process;
then, after standing for 30min, adding phosphate buffer solution with the same volume of 2% FCS into the mixed solution in the second centrifuge tube to stop the digestion of the enzyme, and detecting whether the tissue is completely digested, if the tissue is completely digested, carrying out the next step; if the digestion is not complete, continuously placing the second centrifuge tube in a water bath kettle of 37 ℃ for 10min, and continuously gently blowing and beating the centrifuge tube by using a suction tube in the process;
and finally, after complete digestion, taking out the second centrifuge tube in the water bath, filtering the mixed solution in the second centrifuge tube by using a filter screen, and pouring the filtered mixed solution into a 15mL third centrifuge tube, wherein the liquid obtained in the third centrifuge tube is the single cell suspension.
Preferably, in step 1, 1mL of the enzyme mixture is prepared by mixing 880mL of Hank's buffer, 100ul of 25U/mL collagenase I, 10ul of 60U/mL hyaluronidase, 10ul of 60U/mL deoxyribonuclease and 10ul of 450U/mL collagenase XI, and 2% FCS may be appropriately added when preparing the enzyme mixture.
Preferably, the aortic blood vessels removed in step 3 comprise the sinuses on the aortic arch, the brachiocephalic artery, the left common carotid artery and the left subclavian artery.
Preferably, the process of cutting the aortic blood vessels by using the ophthalmic scissors in the step 5 is controlled within 10 min.
Compared with the prior art, the invention has the beneficial effects that:
1. the method for preparing the mouse vascular tissue single cell suspension by using the shearing and mixed enzyme combined digestion method is simple, convenient and feasible, has more immune cells, and provides an effective tissue single cell suspension preparation method for the research of vascular immune cells.
2. Compared with mechanical shearing and grinding of vascular tissues, the technical scheme has the advantages of high yield of single cells, high activity, strong function, sufficient cell quantity, less cell fragments and lighter cell adhesion and agglomeration phenomenon, and lays a foundation for the later-stage flow detection of vascular immune cells.
3. Since cells are easy to die away from the tissue, the whole process is soaked in 2% FCS and rapidly performed on ice in order to ensure the viability of the cells, thereby ensuring that the cells have high viability after the operation is completed.
4. The technical scheme combines a mechanical fermentation method and an enzyme digestion method, and utilizes the optimal temperature of enzyme activity and adopts collagen, hyaluronic acid and DNA enzyme for treatment. Through repeated digestion, the problem that vascular tissues are easy to adhere and agglomerate in the operation process can be well treated, and cells with complete forms, good dispersity, clean background, less impurities, different cell sizes and various forms are obtained.
Drawings
FIG. 1 is one of the flow cytometric analysis diagrams;
FIG. 2 is a second diagram of flow cytometry analysis after preparation of cell sap according to the present technique;
FIG. 3 is a diagram showing the results of the detection after the preparation of the cell sap according to the present technical solution.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a technical scheme that: a method for preparing mouse aortic artery cell single cell suspension specifically comprises the following steps:
s01, firstly, preparing 1ml of enzyme mixed liquor, a living healthy wild mouse, an operating platform with a whole desktop being a smooth ice surface and a plurality of instruments;
s02, injecting 200-500 ul chloral hydrate into the abdominal cavity of the prepared wild mouse for anesthesia and death, immediately filling and washing the blood vessel of the wild mouse by using 20ml of phosphate buffer saline solution, integrally sterilizing the dead wild mouse by using alcohol with the volume fraction of 75%, performing thoracoabdominal operation after sterilization is completed, and exposing the heart and the aorta, wherein the whole operation process is completed on the operation table;
s03, then, taking an aorta blood vessel with the length of 11mm from the wild mouse body after the thoracoabdominal operation is completed, putting the aorta blood vessel into a first centrifuge tube with the length of 1.5mL and containing 1mL of 2% FCS solution, completely soaking the aorta blood vessel in the solution, and placing the first centrifuge tube on the operation table after the operation is completed;
s04, rapidly removing fat and connective tissues around the aorta blood vessel soaked in the first centrifuge tube with the aid of a microscope, keeping the aorta blood vessel intact in the operation process, and completing the whole operation process on the operation table;
s05, taking the aorta blood vessel after the separation of fat and connective tissue out of the first centrifuge tube, placing the aorta blood vessel into a second centrifuge tube with the volume of 1.5ml, adding 100ul of the enzyme-mixed digestive juice into the second centrifuge tube, completely soaking the aorta blood vessel in the solution, shearing the aorta blood vessel into fragments by ophthalmic scissors, and completing the whole operation process on the operation table;
s06, washing the inner walls of the ophthalmic scissors and a second centrifuge tube with 900ul of the enzyme mixed solution, covering the second centrifuge tube tightly, putting the second centrifuge tube in a 37-DEG water bath, and placing for 30min, wherein a straw is continuously used for blowing and beating in the process;
s07, after the second centrifugal tube is placed for 30min, adding phosphate buffer solution with the same volume as 2% FCS into the mixed solution in the second centrifugal tube to stop the digestion of the enzyme, detecting whether the tissue is completely digested, and if the tissue is completely digested, carrying out the next step; if the digestion is not complete, continuously placing the second centrifuge tube in a water bath kettle of 37 ℃ for 10min, and continuously gently blowing and beating the centrifuge tube by using a suction tube in the process;
and S08, finally, taking out the second centrifuge tube placed in the water bath kettle after complete digestion, filtering the mixed liquid in the second centrifuge tube through a filter screen, and pouring the filtered mixed liquid into a 15mL third centrifuge tube, wherein the liquid obtained in the third centrifuge tube is the single cell suspension.
In step 1, 1mL of the enzyme mixture was prepared by mixing 880mL of Hank's buffer, 100ul of 25U/mL collagenase I, 10ul of 60U/mL hyaluronidase, 10ul of 60U/mL DNAse, and 10ul of 450U/mL collagenase XI, and 2% FCS was added as appropriate when preparing the enzyme mixture.
The aorta vessel taken down in the step 3 comprises a sinus on an aortic arch, the aortic arch, a brachiocephalic artery, a left common carotid artery and a left subclavian artery.
In the step 5, the process of cutting the aorta blood vessel by using the ophthalmic scissors is controlled within 10 min.
In another embodiment, the method for preparing the mouse aortic artery cell single cell suspension can be further realized by the following steps, and specifically comprises the following steps:
s10, firstly, preparing 1ml of enzyme mixed liquor, a living healthy wild mouse, an operating platform with a whole desktop being a smooth ice surface, a plurality of instruments and 1ml of the enzyme mixed liquor;
s20, then, placing the stripped aorta blood vessel into a first digestion tube, dropwise adding 100ul of enzyme mixed liquor into the first digestion tube, and cutting the aorta blood vessel in the first digestion tube into pieces, wherein the whole process is carried out on ice, and the time is controlled within 10 min;
s30, adding 900ul of enzyme mixed solution into the first digestion tube, and fully mixing the enzyme mixed solution with the cut aorta blood vessel;
s40, covering the first digestion tube tightly and placing the first digestion tube in a 37-degree water bath kettle completely for incubation for 40min, and continuously shaking the first digestion tube in the incubation process until the aortic vascular tissue is completely digested;
s50, centrifuging the digested aortic vascular tissue for a short time at 4 ℃ for 5min to finish centrifugation, wherein the centrifugation temperature is 800 ℃;
s60, mixing the centrifuged aortic vascular tissue uniformly by using a suction tube, filtering the mixture into a 15ml centrifuge tube, washing the first digestion tube by using 1ml 2% FCS (FCS-based solvent), and adding the solution into the centrifuge tube after washing;
s70, then, centrifuging the centrifuge tube again, wherein the centrifugal force value is 800 at 4 ℃, the time is 5min, and repeating the previous step when the centrifugation is finished;
s80, and finally, filtering the centrifugate to obtain the single cell suspension.
The enzyme mixture in step 1 comprises collagenase I, hyaluronidase, collagenase XI and DNase.
The working principle is as follows: analyzing and sorting cell or subcellular structure by flow cytometry, soaking the tissue in 2% FCS, completing the whole operation process on ice surface, maintaining cell activity at low temperature, taking out blood vessel, stripping off fat and connective tissue to avoid the toxic action of fat on cell and raise cell activity, combining mechanical process and enzyme digestion process, controlling water bath temperature at 37 deg.c to make enzyme activity at active temperature, and adding collagen, hyaluronic acid and DNA enzyme to speed the digestion of elastic tissue and connective tissue and save time. The problem that the vascular tissues are easy to adhere and agglomerate in the treatment process is solved by adopting a repeated digestion mode, so that the single cells with high yield, high activity and strong functions are obtained.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (4)
1. A method for preparing mouse aortic artery cell single cell suspension is characterized in that: the method specifically comprises the following steps:
s01, firstly, preparing 1ml of enzyme mixed liquor, a living healthy wild mouse, an operating platform with a whole desktop being a smooth ice surface and a plurality of instruments;
s02, injecting 200-500 ul chloral hydrate into the abdominal cavity of the prepared wild mouse for anesthesia and death, immediately filling and washing the blood vessel of the wild mouse by using 20ml of phosphate buffer saline solution, integrally sterilizing the dead wild mouse by using alcohol with the volume fraction of 75%, performing thoracoabdominal operation after sterilization is completed, and exposing the heart and the aorta, wherein the whole operation process is completed on the operation table;
s03, then, taking an aorta blood vessel with the length of 11mm from the wild mouse body after the thoracoabdominal operation is completed, putting the aorta blood vessel into a first centrifuge tube with the length of 1.5mL and containing 1mL of 2% FCS solution, completely soaking the aorta blood vessel in the solution, and putting the first centrifuge tube on the operation table after the operation is completed;
s04, rapidly removing fat and connective tissues around the aorta blood vessel soaked in the first centrifuge tube with the aid of a microscope, keeping the aorta blood vessel intact in the operation process, and completing the whole operation process on the operation table;
s05, taking the aorta blood vessel after the separation of fat and connective tissue out of the first centrifuge tube, placing the aorta blood vessel into a second centrifuge tube with the volume of 1.5ml, adding 100ul of the enzyme-mixed digestive juice into the second centrifuge tube, completely soaking the aorta blood vessel in the solution, shearing the aorta blood vessel into fragments by ophthalmic scissors, and completing the whole operation process on the operation table;
s06, washing the inner walls of the ophthalmic scissors and a second centrifuge tube with 900ul of the enzyme mixed solution, covering the second centrifuge tube tightly, putting the second centrifuge tube in a 37-DEG water bath, and placing for 30min, wherein a straw is continuously used for blowing and beating in the process;
s07, after the second centrifugal tube is placed for 30min, adding phosphate buffer solution with the same volume as 2% FCS into the mixed solution in the second centrifugal tube to stop the digestion of the enzyme, detecting whether the tissue is completely digested, and if the tissue is completely digested, carrying out the next step; if the digestion is not complete, continuously placing the second centrifuge tube in a water bath kettle of 37 ℃ for 10min, and continuously gently blowing and beating the centrifuge tube by using a suction tube in the process;
and S08, finally, taking out the second centrifuge tube placed in the water bath kettle after complete digestion, filtering the mixed liquid in the second centrifuge tube through a filter screen, and pouring the filtered mixed liquid into a 15mL third centrifuge tube, wherein the liquid obtained in the third centrifuge tube is the single cell suspension.
2. The method for preparing single cell suspension of mouse aortic artery cells as claimed in claim 1, wherein: in step 1, 1mL of the enzyme mixture was prepared by mixing 880mL of Hank's buffer, 100ul of 25U/mL collagenase I, 10ul of 60U/mL hyaluronidase, 10ul of 60U/mL DNAse, and 10ul of 450U/mL collagenase XI, and 2% FCS was added as appropriate when preparing the enzyme mixture.
3. The method for preparing single cell suspension of mouse aortic artery cells as claimed in claim 1, wherein: the aorta vessel taken down in the step 3 comprises a sinus on an aortic arch, the aortic arch, a brachiocephalic artery, a left common carotid artery and a left subclavian artery.
4. The method for preparing single cell suspension of mouse aortic artery cells as claimed in claim 1, wherein: in the step 5, the process of cutting the aorta blood vessel by using the ophthalmic scissors is controlled within 10 min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114591883A (en) * | 2021-07-20 | 2022-06-07 | 中国人民解放军陆军军医大学第一附属医院 | Preparation method of single cell suspension of aortic cells |
| CN114958717A (en) * | 2022-04-06 | 2022-08-30 | 中山大学附属第一医院 | Method for quickly separating living cells from trace calcified blood vessels |
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| WO2019100044A1 (en) * | 2017-11-20 | 2019-05-23 | Icahn School Of Medicine At Mount Sinai | Inhibiting trained immunity with a therapeutic nanobilogic composition |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114591883A (en) * | 2021-07-20 | 2022-06-07 | 中国人民解放军陆军军医大学第一附属医院 | Preparation method of single cell suspension of aortic cells |
| CN114591883B (en) * | 2021-07-20 | 2024-04-30 | 中国人民解放军陆军军医大学第一附属医院 | Preparation method of aortic cell single-cell suspension |
| CN114958717A (en) * | 2022-04-06 | 2022-08-30 | 中山大学附属第一医院 | Method for quickly separating living cells from trace calcified blood vessels |
| CN114958717B (en) * | 2022-04-06 | 2023-03-10 | 中山大学附属第一医院 | Method for quickly separating living cells from trace calcified blood vessels |
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