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Merck

An optimized protocol for the isolation of rare stromal cell populations from the mouse spleen.

STAR protocols (2023-01-04)
Yannick O Alexandre, Scott N Mueller
ABSTRACT

Lymphoid tissue stromal cells are important regulators of spleen homeostasis and immune responses. Here, we present an optimized protocol that describes the digestion and enrichment steps for the isolation and analysis of rare populations of stromal cells, including fibroblastic reticular cells, perivascular cells, and glial cells found in the spleen. This protocol is suitable for subsequent analysis of spleen stromal cells by flow cytometry or single-cell RNA sequencing and to analyze different disease models. For complete details on the use and execution of this protocol, please refer to Alexandre et al. (2022).1.

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Roche
Collagenasi D, from Clostridium histolyticum
Sigma-Aldrich
Desossiribonucleasi I, lyophilized powder, Protein ≥85 %, ≥400 Kunitz units/mg protein
Sigma-Aldrich
Sieroalbumina, heat shock fraction, pH 7, ≥98%
Sigma-Aldrich
Acido etilendiamminotetraacetico, suitable for electrophoresis, Molecular Biology, 99.0-101.0% (titration)
Sigma-Aldrich
DAPI, for nucleic acid staining
Roche
Dispase® II (neutral protease, grade II), lyophilized, from bacterial, Roche, pkg of 5 × 1 g