title: Tissue Digestion for Stromal Cell and Leukocyte Isolation.
abstract: Tissue mechanical disruption is often not sufficient to disrupt cell-to-cell interactions; this is particularly relevant for stromal cells that are embedded within the extracellular matrix. For this reason, different enzyme combinations have been described to enable the isolation of single-cell populations, particularly stromal cells. This chapter aims to describe different methods used for enzymatic digestion of stromal cell and leukocyte populations from secondary and tertiary lymphoid organs. Collagenase D and P and collagenase D and dispase protocols provide a good yield of stromal cells, while a collagenase dispase-only protocol should be used if the main aim of the technique is to retrieve leukocyte populations. However, for isolation of both stroma and leukocyte populations the collagenase D and P protocol would provide the best results. Protocols for these techniques and illustrative results from flow cytometry analysis can be found in this chapter.
authors.affiliation: Centre for Translational Inflammation Research, Institute of Inflammation and Ageing, College of Medical & Dental Sciences, University of Birmingham Research Laboratories, Queen Elizabeth Hospital, Birmingham, B15 2WB, UK.
authors.forename: Saba
authors.initials: S
authors.lastname: Nayar
authors.affiliation: Centre for Translational Inflammation Research, Institute of Inflammation and Ageing, College of Medical & Dental Sciences, University of Birmingham Research Laboratories, Queen Elizabeth Hospital, Birmingham, B15 2WB, UK.
authors.forename: Joana
authors.initials: J
authors.lastname: Campos
authors.affiliation: Centre for Translational Inflammation Research, Institute of Inflammation and Ageing, College of Medical & Dental Sciences, University of Birmingham Research Laboratories, Queen Elizabeth Hospital, Birmingham, B15 2WB, UK.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Steinthal
authors.affiliation: Centre for Translational Inflammation Research, Institute of Inflammation and Ageing, College of Medical & Dental Sciences, University of Birmingham Research Laboratories, Queen Elizabeth Hospital, Birmingham, B15 2WB, UK. f.barone@bham.ac.uk.
authors.forename: Francesca
authors.initials: F
authors.lastname: Barone
mesh_terms: D000818:Animals; D002450:Cell Communication / Q000502:physiology; D002469:Cell Separation / Q000379:methods; D017364:Collagenases / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D007962:Leukocytes / Q000378:metabolism*; D051379:Mice; D017154:Stromal Cells / Q000378:metabolism*
chemical_list: D017364:Collagenases
keywords: Collagenases; Gut; Leukocytes; Lymph nodes; Salivary glands; Stromal cells; Tissue digestion