Best Practices for Preparing a Single Cell Suspension from Solid Tissues for Flow Cytometry
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Preparing a single cell suspension is a critical step in any solid tissue flow cytometry experiment. Tissue dissection, enzymatic digestion, and mechanical dissociation are three significant steps leading to the degradation of the extracellular matrix and the isolation of single cells, allowing the generation of high‐quality flow cytometry data. Cells and the extracellular matrix contain various proteins and other structures which must be considered when designing a tissue digestion protocol to preserve the viability of cells and the presence of relevant antigens while digesting matrix components and cleaving cell–cell junctions. Evaluation of the single cell suspension is essential before proceeding with the labeling of the cells as high viability and absence of cell debris and aggregates are critical for flow cytometry. The information presented should be used as a general guide of steps to consider when preparing a single cell suspension from solid tissues for flow cytometry experiments. © 2018 International Society for Advancement of Cytometry
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Enzymatic digestion causes substantial loss of some cell-surface receptors.
“Of note, we observed substantial loss of some cell-surface receptors following enzymatic digestion, in line with previous work in peripheral immunology (Supplementary Fig. 1d–f and Supplementary Note 1)3,5–9.”
From Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain (2022) · cites this paper as [9]
The Tissue Disaggregation > Enzymes section says dispase can cleave specific cell-surface molecules or antigens relevant to T-cell analysis. The Enzymatic and Mechanical Dissociation section also warns that strong or concentrated enzymes may compromise surface markers. These passages support loss of some markers, but do not establish that the loss is substantial or occurs with enzymatic digestion generally. (LLM, conf=0.84)
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- Authors
- Reichard, A., & Asosingh, K.
- Affiliations
- Cleveland Clinic (US)
- Corresponding author
- Asosingh, Kewal
- Venue
- Cytometry Part A
- Publisher
- Wiley
- Published
- 2018-12-06
- Volume
- 95
- Issue
- 2
- Pages
- 219-226
- ISSN
- 1552-4922 (ISSN-L)
- 1552-4930
- PMC
- PMC6375754
- OpenAlex
- W2904043980
- Cited by
- 201
- References
- 46
- Citations by year
- 2026: 20 · 2025: 37 · 2024: 27 · 2023: 41 · 2022: 36 · 2021: 23 · 2020: 14 · 2019: 3
- FWCI
- 4.09
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Cytometry Part A (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institutes of Health (HL103453, HL081064, and HL109250)
- Topics
- Physical Sciences › Engineering › Biomedical Engineering › Microfluidic and Bio-sensing Technologies
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
- Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
- Keywords
- single-cell suspension preparation, solid tissue, enzymatic digestion, mechanical dissociation, extracellular matrix degradation, tissue dissection, cell viability, cell-cell contact, cell labeling, flow cytometry sample preparation
- MeSH terms
- Animals, Antigens, Cell Separation, Cell Survival, Extracellular Matrix, Flow Cytometry, Proteins, Single-Cell Analysis
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