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Protocol for isolating stromal cells from lymphoid tissue for performing scRNA-seq.

Spyridon Makris1, Daniel Shewring1, Agnesska C Benjamin1

  • 1Stromal Immunology Group, Laboratory for Molecular Cell Biology, University College London, Gower Street, WC1E 6BT London, UK.

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Researchers developed a new protocol to isolate fibroblasts from lymphoid tissues. This method aids in single-cell RNA sequencing (scRNA-seq) analysis of these rare cells, applicable to tumors too.

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Area of Science:

  • Immunology
  • Cell Biology
  • Biotechnology

Background:

  • Stromal cells, including fibroblasts, are rare (<2%) in lymph nodes.
  • Isolating and analyzing these cells for single-cell RNA sequencing (scRNA-seq) is challenging.
  • Understanding stromal cell function requires effective isolation techniques.

Purpose of the Study:

  • To present a detailed protocol for purifying fibroblasts from lymphoid tissues.
  • To enable downstream single-cell RNA sequencing (scRNA-seq) analysis of stromal cells.
  • To provide a method applicable to various tissues, including tumors.

Main Methods:

  • Lymph node dissection and enzymatic digestion.
  • Flow cytometry for cell staining and viability assessment.
  • Magnetic-activated cell sorting (MACS) for fibroblast purification.

Main Results:

  • A reproducible protocol for isolating fibroblasts from lymphoid tissues was established.
  • The protocol includes quality control steps for purity and viability assessment.
  • Demonstrated applicability to other tissues, such as tumors.

Conclusions:

  • The presented protocol effectively purifies fibroblasts from lymphoid tissues for scRNA-seq.
  • This method overcomes technical difficulties in isolating rare stromal cells.
  • The protocol has broad applicability in immunological and cancer research.