T-Cell Trafficking

Saba Nayar Ph.D., Joana Campos Ph.D., Nathalie Steinthal B.Sc., M.Sc., Francesca Barone M.D., Ph.D., Saba Nayar, Joana Campos, Nathalie Steinthal, Francesca Barone

George Edward Rainger, Helen M. Mcgettrick

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.