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

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Ethylenediaminetetraacetic acid disodium salt dihydrate, suitable for electrophoresis, Molecular Biology, 99.0-101.0% (titration)
Roche
Collagenase D, from Clostridium histolyticum
Roche
Dispase® II (neutral protease, grade II), lyophilized, from bacterial, Roche, pkg of 5 × 1 g
Sigma-Aldrich
DAPI, for nucleic acid staining
Sigma-Aldrich
Deoxyribonuclease I from bovine pancreas, lyophilized powder, Protein ≥85 %, ≥400 Kunitz units/mg protein