title: Unbiased analysis, enrichment and purification of thymic stromal cells.
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.
authors.affiliation: Monash Immunology and Stem Cell Laboratories, Level 3, STRIP, Building 75, Monash University, Wellington Road, Clayton, Victoria, 3800, Australia.
authors.forename: Daniel H D
authors.initials: DH
authors.lastname: Gray
authors.forename: Anne L
authors.initials: AL
authors.lastname: Fletcher
authors.forename: Maree
authors.initials: M
authors.lastname: Hammett
authors.forename: Natalie
authors.initials: N
authors.lastname: Seach
authors.forename: Tomoo
authors.initials: T
authors.lastname: Ueno
authors.forename: Lauren F
authors.initials: LF
authors.lastname: Young
authors.forename: Jade
authors.initials: J
authors.lastname: Barbuto
authors.forename: Richard L
authors.initials: RL
authors.lastname: Boyd
authors.forename: Ann P
authors.initials: AP
authors.lastname: Chidgey
mesh_terms: D000818:Animals; D002469:Cell Separation; D004847:Epithelial Cells / Q000737:chemistry / Q000276:immunology; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000032:analysis / Q000235:genetics; D018189:Immunomagnetic Separation; D007633:Keratins / Q000032:analysis; D017493:Leukocyte Common Antigens / Q000032:analysis; D051379:Mice; D008810:Mice, Inbred C57BL; D010641:Phenotype; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000737:chemistry / Q000276:immunology; D013696:Temperature; D013950:Thymus Gland / Q000737:chemistry / Q000166:cytology* / Q000276:immunology
chemical_list: D051858:Forkhead Transcription Factors; C090140:Whn protein; D007633:Keratins; D017493:Leukocyte Common Antigens; C516770:Ptprc protein, mouse