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Biology

Preparation of a Single-Cell Suspension from Mouse Carotid Arteries for Single-Cell Sequencing

Published: January 19th, 2024

DOI:

10.3791/65863

1Cyrus Tang Hematology Center, Cyrus Tang Medical Institute, Soochow University, 2Collaborative Innovation Center of Hematology of Jiangsu Province, Soochow University, 3Suzhou Key Laboratory of Thrombosis and Vascular Biology, Soochow University

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 ....

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All animal procedures described below were approved by the Institutional Animal Care and Use Committee of Soochow University.

1. Reagents and materials preparation

  1. Utilize 1x PBS without calcium and magnesium and 2.5 U/mL heparin sodium salt to prepare the perfusion solution. Store at 4 °C, and precool on ice when used.
  2. Prepare dissociation reagent A that contains 125 CDU/mL collagenase II and 60 U/mL DNase I by diluting 1250 CDU/mL collagenase II an.......

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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.......

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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.......

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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.).

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Name Company Catalog Number Comments
0.25% EDTA-free trypsin Beyotime C0205 Dilute 1 mL of 0.25% EDTA-free trypsin into 1 mL of 1x PBS.
0.9% NaCl saline solution Beyotime ST341 Dilute the heparin sodium solution into a final concentration of 10 mg/mL
1 mL syringes  SKJYLEAN sk-r009 To perform cardiac perfusion
1.5 mL centrifuge tubes KIRGEN KG2211W To centrifuge the tissue piece and cell suspension
20 mL syringes SKJYLEAN sk-r013 To perform cardiac perfusion
40 µm cell strainer JETBIOFIL css010040 To filter undigested tissue fragments
AO/PI kit Hengrui Biological RE010212 To identify whether the cell is alive or dead
Automated cell counter Countstar Mira FL To analyze the cell morphology and cell viability of digested carotid vascular cells
Cell Ranger software 10× Genomics 3.0.2 To process Chromium single-cell RNA-seq output and perform clustering and gene expression analysis
Chromium Single Cell 3'Reagent Kits v3 10× Genomics 1000075 To prepare single-cell RNA-seq libraries of single-cell suspension
Collagenase II Sigma-Aldrich C6885 Dilute with HBSS to a final concentration of 125 CDU/mL
Deoxyribonuclease I Worthington LS002140 Dilute with HBSS to a final concentration of 60 U/mL
Fetal bovine serum  HyClone SH30088.03 Termination of the digestion reaction
Hank's balanced salt solution  Gibco 14175095 Store at the room temperture
Heparin sodium salt Solarbio Life Science H8060 Dilute with 0.9% NaCl to a final concentration of 10 mg/mL
Microcentrifuge Thermo Fisher Scientific 75002560 Applied for spining down the tissues and cell pellets
NovaSeq 6000  Illumina N/A Sequencer
Phosphate-buffered saline Solarbio Life Science P1000 Used for cardiac perfusion and resuspension of cells
Seurat package- R Satija Lab 3.1.2 To performed dimensionality reduction, visualization, and analysis of scRNA-sequencing data
Six-well cell culture plates NEST 703002 Place the vascular tissue
Water bath Jinghong DK-S22 Keep the digestion temperature at 37 °C

  1. Lee, D. -. Y., Chiu, J. -. J. Atherosclerosis and flow: roles of epigenetic modulation in vascular endothelium. Journal of Biomedical Science. 26 (1), 56 (2019).
  2. Libby, P., et al. Atherosclerosis. Nature Reviews Disease Primers. 5 (1), 56 (2019).
  3. Jovic, D., et al. Single-cell RNA sequencing technologies and applications: A brief overview. Clinical and Translational Medicine. 12 (3), e694 (2022).
  4. Li, F., et al. Single-cell RNA-seq reveals cellular heterogeneity of mouse carotid artery under disturbed flow. Cell Death Discovery. 7 (1), 180 (2021).
  5. Rohlenova, K., et al. Single-cell RNA sequencing maps endothelial metabolic plasticity in pathological angiogenesis. Cell Metabolism. 31 (4), 862.e14-877.e14 (2020).
  6. Ren, X., et al. Insights gained from single-cell analysis of immune cells in the tumor microenvironment. Annual Review of Immunology. 39, 583-609 (2021).
  7. Sun, H., Xu, X., Deng, C. Preparation of single epithelial cells suspension from mouse mammary glands. Bio-Protocol. 10 (4), e3530 (2020).
  8. Korin, B., Chung, J. -. J., Avraham, S., Shaw, A. S. Preparation of single-cell suspensions of mouse glomeruli for high-throughput analysis. Nature Protocols. 16 (8), 4068-4083 (2021).
  9. Thomson, B., Quaggin, S. Preparation of a single cell suspension from the murine iridocorneal angle. Bio-Protocol. 12 (10), e4426 (2022).
  10. Leale, D. M., et al. A two-stage digestion of whole murine knee joints for single-cell RNA sequencing. Osteoarthritis and Cartilage Open. 4 (4), 100321 (2022).
  11. Hu, D., Yin, C., Mohanta, S. K., Weber, C., Habenicht, A. J. R. Preparation of single-cell suspensions from mouse aorta. Bio-Protocol. 6 (11), e1832 (2016).
  12. Jungblut, M., Oeltze, K., Zehnter, I., Hasselmann, D., Bosio, A. Standardized preparation of single-cell suspensions from mouse lung tissue using the gentleMACS dissociator. Journal of Visualized Experiments. 29, e1266 (2009).
  13. You, Y., et al. Benchmarking UMI-based single-cell RNA-seq preprocessing workflows. Genome Biology. 22 (1), 339 (2021).
  14. Stuart, T., et al. Comprehensive integration of single-cell data. Cell. 177 (7), 1888-1902 (2019).
  15. Depuydt, M. A. C., et al. Microanatomy of the human atherosclerotic plaque by single-cell transcriptomics. Circulation Research. 127 (11), 1437-1455 (2020).
  16. Gao, X. -. F., et al. Single-cell RNA sequencing of the rat carotid arteries uncovers potential cellular targets of neointimal hyperplasia. Frontiers in Cardiovascular Medicine. 8, 75152 (2021).
  17. Kumar, S., et al. Isolation of endothelial cells from the lumen of mouse carotid arteries for single-cell multi-omics experiments. Journal of Visualized Experiments. 176, e63128 (2021).

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