CN115141792A - Preparation method and application of mouse carotid artery blood vessel tissue single cell suspension - Google Patents
Preparation method and application of mouse carotid artery blood vessel tissue single cell suspension Download PDFInfo
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Abstract
The invention discloses a preparation method of a mouse carotid artery blood vessel tissue single cell suspension, which comprises the steps of shearing mouse carotid artery tissue, adding dissociation working solution RA for digestion for 1 hour, then centrifuging to remove supernatant, adding dissociation working solution RB, and finally centrifuging to obtain the high-quality single cell suspension. The invention also provides an application of the preparation method in the related fields of single cell sequencing and flow detection. The invention has the beneficial effects that: the invention provides a preparation method and application of mouse carotid artery blood vessel tissue single cell suspension for single cell sequencing, and particularly relates to single cell sequencing by using various tissue digestive enzymes to carry out stepwise digestion on mouse carotid artery tissue so as to control the tissue dissociation degree to obtain high-quality single cell suspension, thereby greatly increasing the feasibility and accuracy of single cell sequencing and flow detection technology, promoting the clinical diagnosis and theoretical mechanism research of various diseases, and having important application prospect and popularization value.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a preparation method and application of a mouse carotid vascular tissue single-cell suspension.
Background
In recent years, with the rapid development of sequencing technologies, single-cell transcriptome sequencing technologies such as 10 × Genomics for revealing cell heterogeneity have come into force. The method carries out high-throughput sequencing and analysis on RNA at the single cell level, detects the expression conditions of all genes in the whole genome range of cells, and is applied to the fields of tumors, neural development, cardiovascular diseases, immunology and the like to a certain extent. However, because different tissues have different physiological characteristics, when a conventional dissociation method is used to prepare a single cell suspension, various problems that the single cell capture is affected by low cell survival rate, small cell number, more cell fragments and the like often occur. Therefore, the invention aims at the digestion method of different tissues and has important significance in the research fields of tumor, cardiovascular disease and the like when preparing high-quality single cell suspension.
At present, the preparation of single cell suspension of mouse vascular tissue by collagenase or elastase is often performed by enzymatic digestion. It degrades the elastic fiber and collagen fiber in the tissue to destroy the protein responsible for tight connection between cells, so as to dissociate the cells. However, different tissues have different physiological characteristics, and conventional invention patents such as CN111621466A and CN107748130A are directed to single cell suspension preparation of mouse heart and pulmonary artery tissues, and no digestion method specific to mouse carotid artery vascular tissues has been reported.
The prior art often adopts a single dissociation mode and is difficult to play a good role in the digestion of mouse carotid artery tissues. The invention discloses a method for stepwise digesting by using various types of enzymes, which is beneficial to obtaining high-quality single cell suspension, thereby greatly increasing the feasibility and the accuracy of single cell sequencing and flow detection technologies and promoting the clinical diagnosis and theoretical mechanism research of various diseases.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides a preparation method and application of a mouse carotid artery blood vessel tissue single cell suspension. The invention is realized by the following technical scheme.
A preparation method of a mouse carotid artery blood vessel tissue single cell suspension comprises the following steps:
s1: after anesthetizing the mice, performing heart perfusion by using precooled PBS (phosphate buffer solution), and removing tissues around carotid arteries;
s2: longitudinally dissecting the carotid artery, placing in a six-well plate containing 1.5% FBS;
s3: each carotid blood vessel was cut to approximately 2mm with surgical scissors 2 The 4 segments of (1.5 mL) are aspirated into a 1.5mL EP tube and centrifuged at 2000rpm for 5 minutes to pellet the vascular tissue;
s4: preparing 1mL of tissue dissociation RA working solution by using HBSS solution;
s5: adding 500 mu L of dissociation working solution into each tube, and putting the tube into a water bath kettle at 37 ℃ for heating and dissociation for 1 hour;
s6: blowing and beating once by using a 1mL liquid transfer gun every 10 minutes;
s7:1 hours later, 500. Mu.L of 5-FBS was added thereto and mixed, followed by filtration using a 40 μm filter;
s8: placing the mixture into a centrifuge, and centrifuging the mixture for 5 minutes at 1000 rpm;
s9: absorbing and removing the supernatant, and adding 200 mu L of tissue dissociation RB working solution;
s10: digesting in a water bath kettle at 37 ℃ for 5 minutes, and lightly blowing and beating once every 1 minute;
s11: digestion was terminated using 200 μ L of 5% FBS, centrifuged at 1000rpm for 5 minutes;
s12: the supernatant was aspirated and resuspended in 100. Mu.L PBS;
s13: cell concentration and viability in cell suspensions were measured using a cytometer.
Preferably, the tissue dissociation RA working solution in the step S4 comprises 1mg/mL type II collagenase and 0.02mg/mL DNase.
Preferably, in the tissue dissociation RB working solution described in step S9, the ratio of HBSS to 0.25% EDTA-free pancreatin is 1.
The invention also provides an application of the preparation method in the related fields of single cell sequencing and flow detection.
Preferably, single Cell transcriptome sequencing is performed on the Single Cell suspension by using 10 XGenomics platform chromosome Single Cell 3' reagent kit v3 reagent, and gene matching analysis is performed on the raw sequencing data by using CellRanger software to obtain high-quality Single Cell sequencing data.
The invention has the beneficial effects that: the invention provides a preparation method and application of mouse carotid artery blood vessel tissue single cell suspension for single cell sequencing, and particularly relates to single cell sequencing by using various tissue digestive enzymes to carry out stepwise digestion on mouse carotid artery tissue so as to control the tissue dissociation degree to obtain high-quality single cell suspension, thereby greatly increasing the feasibility and accuracy of single cell sequencing and flow detection technology, promoting the clinical diagnosis and theoretical mechanism research of various diseases.
Drawings
FIG. 1 is a flow chart of the production process of the present invention;
FIG. 2 shows the cell morphology after digestion;
FIG. 3 is a diagram of the raw data of single cells with high quality obtained after single cell sequencing using the single cell suspension prepared by the present invention.
Detailed Description
The embodiments of the present invention will be described more fully hereinafter.
The invention provides a preparation method and application of mouse carotid artery blood vessel tissue single cell suspension for single cell sequencing, belongs to the technical field of biomedicine, and particularly relates to single cell sequencing by using various types of tissue digestive enzymes to carry out stepwise digestion on mouse carotid artery tissue so as to control the tissue dissociation degree to obtain high-quality single cell suspension.
The complete technical scheme of the invention is as follows:
a preparation method of a mouse carotid artery blood vessel tissue single cell suspension comprises the following steps:
s1: after anesthetizing the mice, performing heart perfusion by using precooled PBS (phosphate buffer solution), and removing tissues around carotid arteries;
s2: the carotid artery was dissected longitudinally and placed in a six-well plate containing 1.5% FBS;
s3: each carotid blood vessel was cut to approximately 2mm with surgical scissors 2 The 4 segments of (4), aspirated into 1.5mL EP tubes, and centrifuged at 2000rpm for 5 minutes to pellet vascular tissues;
s4: preparing 1mL of tissue dissociation RA working solution by using HBSS solution;
s5: adding 500 mu L of dissociation working solution into each tube, and putting the tubes into a water bath kettle at 37 ℃ for heating and dissociation for 1 hour;
s6: blowing and beating once by using a 1mL liquid transfer gun every 10 minutes;
s7: after 1 hour, 500. Mu.L of 5% FBS was added thereto and mixed, followed by filtration using a 40 μm filter;
s8: placing the mixture into a centrifuge, and centrifuging the mixture for 5 minutes at 1000 rpm;
s9: absorbing and removing the supernatant, and adding 200 mu L of tissue dissociation RB working solution;
s10: digesting in a water bath kettle at 37 ℃ for 5 minutes, and lightly blowing and beating once every 1 minute;
s11: digestion was terminated using 200. Mu.L of 5% FBS, and centrifugation was carried out at 1000rpm for 5 minutes;
s12: the supernatant was aspirated and resuspended in 100. Mu.L PBS;
s13: cell concentration and viability in cell suspensions were measured using a cytometer.
Wherein, the first and the second end of the pipe are connected with each other,
the tissue dissociation RA working solution in the step S4 comprises 1mg/mL type II collagenase and 0.02mg/mL DNase;
in the tissue dissociation RB working solution described in step S9, the ratio of HBSS to 0.25% EDTA-free pancreatin was configured to be 1.
The first embodiment is as follows: the invention can prepare high-quality single cell suspension
The technical scheme of the invention is used for digesting the carotid artery vascular tissue of the mouse to prepare single cell suspension. According to the detection of the automatic cell counter, the two groups of carotid vascular tissues in the embodiment are successfully dissociated into single-cell suspensions, the diameters of the cells are uniform, the total amount of the cells is large, the cell viability is detected by using AO/PI double-fluorescence staining, and the primary cell viability digested by using the technical scheme of the invention is up to about 90 percent and meets the quality inspection requirement of single-cell on-machine capture.
The second embodiment: high quality single cell sequencing data can be obtained using single cell suspensions digested by the invention
Furthermore, 10 XGenomics platform chromosome Single Cell 3' reagent kit v3 reagent was used to perform Single Cell transcriptome sequencing on the Single Cell suspension, and the original sequencing data was subjected to gene matching analysis by using CellRanger software, and then the Single Cell sequencing data quality was found to be good. Wherein, the total number of Reads in sequencing reaches more than 64000 ten thousand, the Barcodes effective rate reaches more than 96%, and the average gene factor detected by each cell is more than 2600. Therefore, the single cell suspension prepared by the invention can obtain high-quality single cell sequencing data and has high application value.
The invention is used for dissociating mouse histiocyte;
the invention discloses application of tissue dissociation in the related fields of single cell sequencing and flow detection.
In the description herein, reference to the terms "one embodiment," "some embodiments," "an example," "a specific example," or "some examples" or the like means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the present invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (5)
1. A preparation method of a mouse carotid vascular tissue single cell suspension is characterized by comprising the following steps:
s1: after anesthetizing the mice, performing heart perfusion by using precooled PBS (phosphate buffer solution), and removing tissues around carotid arteries;
s2: longitudinally dissecting the carotid artery, placing in a six-well plate containing 1.5% FBS;
s3: each carotid blood vessel was cut to approximately 2mm with surgical scissors 2 The 4 segments of (4), aspirated into 1.5mL EP tubes, and centrifuged at 2000rpm for 5 minutes to pellet vascular tissues;
s4: preparing 1mL of tissue dissociation RA working solution by using HBSS solution;
s5: adding 500 mu L of dissociation working solution into each tube, and putting the tubes into a water bath kettle at 37 ℃ for heating and dissociation for 1 hour;
s6: blowing and beating once by using a 1mL pipette at intervals of 10 minutes;
s7: after 1 hour, 500. Mu.L of 5% FBS was added thereto and mixed, followed by filtration using a 40 μm filter;
s8: placing the mixture into a centrifuge, and centrifuging the mixture for 5 minutes at 1000 rpm;
s9: after the supernatant was aspirated, 200. Mu.L of tissue dissociation RB working solution was added;
s10: digesting in a water bath kettle at 37 ℃ for 5 minutes, and lightly blowing and beating once every 1 minute;
s11: digestion was terminated using 200 μ L of 5% FBS, centrifuged at 1000rpm for 5 minutes;
s12: the supernatant was aspirated and resuspended in 100. Mu.L PBS;
s13: cell concentration and viability in cell suspensions were measured using a cytometer.
2. The method for preparing the mouse carotid vascular tissue single cell suspension according to claim 1, which is characterized in that: the tissue dissociation RA working solution described in step S4 comprises 1mg/mL collagenase type II and 0.02mg/mL DNase.
3. The method for preparing the mouse carotid artery blood vessel tissue single cell suspension according to the claim 1, which is characterized in that: in the tissue dissociation RB working solution described in step S9, the ratio of HBSS to 0.25% EDTA-free pancreatin was configured to be 1.
4. Use of the preparation method according to any one of claims 1-3 in the fields related to single cell sequencing and flow detection.
5. The use of claim 4, wherein Single Cell transcriptome sequencing of Single Cell suspensions was performed using 10 XGenomics platform chromosome Single Cell 3' reagent Kits v3 reagent, and high quality Single Cell sequencing data was obtained by gene matching analysis of raw sequencing data using CellRanger software.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117384825A (en) * | 2023-11-24 | 2024-01-12 | 杭州联川生物技术股份有限公司 | Enzymolysis liquid for preparing single cell suspension of human arterial tissue, kit, method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104403990A (en) * | 2014-11-06 | 2015-03-11 | 北京农学院 | Method for separating and purifying mouse intestinal mucosa micro-vascular endothelial cells |
| CN112226406A (en) * | 2020-10-19 | 2021-01-15 | 中国医学科学院北京协和医院 | Preparation method of human perivascular adipose tissue single cell suspension |
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- 2022-07-16 CN CN202210837887.4A patent/CN115141792A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104403990A (en) * | 2014-11-06 | 2015-03-11 | 北京农学院 | Method for separating and purifying mouse intestinal mucosa micro-vascular endothelial cells |
| CN112226406A (en) * | 2020-10-19 | 2021-01-15 | 中国医学科学院北京协和医院 | Preparation method of human perivascular adipose tissue single cell suspension |
Non-Patent Citations (2)
| Title |
|---|
| FENGCHAN LI等: "Single-cell RNA-seq reveals cellular heterogeneity of mouse carotid artery under disturbed flow", CELL DEATH DISCOVERY, pages 1 - 14 * |
| 朱玉珍;武文;田野苹;杨继庆;李润平;: "小鼠脑微血管内皮细胞的原代培养与纯化", 解剖学杂志, no. 05, pages 530 - 533 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117384825A (en) * | 2023-11-24 | 2024-01-12 | 杭州联川生物技术股份有限公司 | Enzymolysis liquid for preparing single cell suspension of human arterial tissue, kit, method and application thereof |
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