Unbiased analysis, enrichment and purification of thymic stromal cells

Research output: Contribution to journalArticle

Authors

  • Daniel H D Gray
  • Maree Hammett
  • Natalie Seach
  • Tomoo Ueno
  • Lauren F Young
  • Jade Barbuto
  • Richard L Boyd
  • Ann P Chidgey

Colleges, School and Institutes

Abstract

The microenvironment of the thymus consists of functionally discrete niches composed of distinct stromal cell subsets. Clinically relevant changes affecting T-cell differentiation occur within these niches with age and injury caused by irradiation and chemotherapy treatments. The study of thymic stromal cells has been hampered by the technical difficulty in isolating significant numbers of this important population. Here we present an improved protocol for enzymatic isolation of stromal cells that enables comparative flow cytometric analyses and their purification for downstream cellular or molecular analysis. Fractions analyzed throughout enzymatic digestion of the thymus revealed that various stromal subsets are isolated at characteristic intervals. This highlights the importance of pooling all cells isolated from the thymus for numerical and phenotypic analysis to avoid biased representation of subpopulations. We also describe refined magnetic bead separation techniques that yield almost pure preparations of CD45(-) stroma. Sorting of these suspensions using defined markers enabled purification of the major epithelial subsets, confirmed by keratin staining and PCR analysis. This three-step procedure represents a rapid, reproducible method for the unbiased purification of the stromal cells that direct thymic T-cell differentiation.

Details

Original languageEnglish
Pages (from-to)56-66
Number of pages11
JournalJournal of Immunological Methods
Volume329
Issue number1-2
Publication statusPublished - 1 Jan 2008

Keywords

  • Animals, Antigens, CD45, Cell Separation, Epithelial Cells, Flow Cytometry, Forkhead Transcription Factors, Immunomagnetic Separation, Keratins, Mice, Mice, Inbred C57BL, Phenotype, Reproducibility of Results, Reverse Transcriptase Polymerase Chain Reaction, Stromal Cells, Temperature, Thymus Gland