Published: January 19th, 2024
Here we describe a two-step cell digestion protocol for preparing a single-cell suspension of mouse carotid arteries.
Carotid arteries are major blood vessels in the neck that supply blood and oxygen to the brain, but carotid stenosis occurs when carotid arteries are clogged by plaque. Revealing the cellular composition of the carotid artery at the single-cell level is essential for treating carotid atherosclerosis. However, there is no ready-to-use protocol for the preparation of single-cell suspensions from carotid arteries. To obtain a suitable protocol for the dissociation of normal carotid arteries at the single-cell level with less damage to cells, we designed a two-step digestion method by integrating the digestion process of collagenase/DNase and trypsin. Acridine orange/propidium iodide (AO/PI) dual-fluorescence counting was used to detect cell viability and concentration, and it was found that the single-cell suspension satisfied the requirements for single-cell sequencing, with the viability of cells over 85% and a high cell concentration. After single-cell data processing, a median of ~2500 transcripts per cell were detected in each carotid artery cell. Notably, a variety of cell types of the normal carotid artery, including vascular smooth muscle cells (VSMCs), fibroblasts, endothelial cells (ECs), and macrophages and dendritic cells (Mφ/DCs), were concurrently detectable. This protocol may be applied to prepare a single-cell suspension of blood vessels from other tissues with appropriate modifications.
Atherosclerosis is a chronic inflammatory disease associated with risk factors such as high blood pressure, hyperlipidemia, and hemodynamics1. Carotid artery bifurcations are prone to hemodynamic changes and lead to carotid plaque formation. The clinical presentation of carotid atherosclerosis can be acute such as stroke and transient cerebral ischemia, or chronic such as recurrent transient cerebral ischemia and vascular dementia2. Mechanically, carotid plaque is the outcome of the interaction between different vessel wall cells and various blood cells under pathological conditions. Therefore, revealing the single-cell ....
All animal procedures described below were approved by the Institutional Animal Care and Use Committee of Soochow University.
1. Reagents and materials preparation
This protocol describes a two-step cell digestion method for preparing a single-cell suspension of mouse carotid arteries (Figure 1). This two-step cell digestion method combines collagenase/DNase with trypsin to effectively dissociate the mouse carotid vascular wall to obtain high-quality single-cell suspensions for single-cell sequencing. After dissociation, the cell concentration and cell viability were calculated by an automated cell counter. The bright field showed the morphology of car.......
Here we provide a detailed protocol for the preparation of a high-quality single-cell suspension from the carotid artery of wild-type mice, in which a two-step digestion method integrating the digestion process of collagenase/DNase and trypsin was constructed. After the quality check of the single-cell suspension, we found that it satisfied the requirements for single-cell sequencing, with the viability of cells over 85% and a high cell concentration. Moreover, a variety of cell types in the carotid artery, including VSM.......
This work was supported by grants from the Natural Science Foundation of China (82070450 to C.T.and 82170466 to L.Z.) and the fellowship of China Postdoctoral Science Foundation (7121102223 to F.L.).....
|0.25% EDTA-free trypsin
|Dilute 1 mL of 0.25% EDTA-free trypsin into 1 mL of 1x PBS.
|0.9% NaCl saline solution
|Dilute the heparin sodium solution into a final concentration of 10 mg/mL
|1 mL syringes
|To perform cardiac perfusion
|1.5 mL centrifuge tubes
|To centrifuge the tissue piece and cell suspension
|20 mL syringes
|To perform cardiac perfusion
|40 µm cell strainer
|To filter undigested tissue fragments
|To identify whether the cell is alive or dead
|Automated cell counter
|To analyze the cell morphology and cell viability of digested carotid vascular cells
|Cell Ranger software
|To process Chromium single-cell RNA-seq output and perform clustering and gene expression analysis
|Chromium Single Cell 3'Reagent Kits v3
|To prepare single-cell RNA-seq libraries of single-cell suspension
|Dilute with HBSS to a final concentration of 125 CDU/mL
|Dilute with HBSS to a final concentration of 60 U/mL
|Fetal bovine serum
|Termination of the digestion reaction
|Hank's balanced salt solution
|Store at the room temperture
|Heparin sodium salt
|Solarbio Life Science
|Dilute with 0.9% NaCl to a final concentration of 10 mg/mL
|Thermo Fisher Scientific
|Applied for spining down the tissues and cell pellets
|Solarbio Life Science
|Used for cardiac perfusion and resuspension of cells
|Seurat package- R
|To performed dimensionality reduction, visualization, and analysis of scRNA-sequencing data
|Six-well cell culture plates
|Place the vascular tissue
|Keep the digestion temperature at 37 °C
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