Tissue Digestion for Stromal Cell and Leukocyte Isolation.

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.