Dissociation of Murine Mammary Gland and Tumor Tissues to Create Highly Viable Single-Cell Suspensions

The dissociation of tissue into single-cell suspensions for analysis aids in understanding what is happening at the cellular level in the tumor microenvironment. A longstanding challenge in obtaining and utilizing primary cells is producing high-quality cell suspensions that preserve cell characteristics for downstream processing, including single-cell RNA sequencing (scRNA-Seq) or immunophenotyping via flow cytometry. Typically, tissues are dissociated using a combination of mechanical disruption, enzymatic digestion, and filtration, and several factors in each step introduce variability into the resulting product. Therefore, optimization steps must be taken to identify a method that works best for any specific tissue type. For example, mammary fat pad tissue is rich in adipocytes, making it more challenging to obtain a clean cell suspension and to successfully preserve viable cells. As a result, generating comparable cell suspensions from mammary fat pad tissue and epithelioid tumors is challenging. This protocol establishes a reproducible, cost-effective method for generating highly viable single-cell suspensions from murine mammary gland and tumor tissues that preserve cell-surface epitopes for further analysis. This procedure has been used to produce high-quality scRNA-Seq libraries and to perform immunophenotyping using multi-parameter flow cytometry.