Protocol for Single-Cell Dissociation, RNA Sequencing, and Isolation of Myoepithelial Cells and Fibroblasts from the Aged Lacrimal and Salivary Glands.

Aging of the lacrimal gland (LG) is associated with reduced tear secretion and chronic inflammation, contributing to dry eye disease. Age-related LG changes include immune cell infiltration, cellular damage, lipid accumulation in acinar cells, extracellular matrix deposition, and ductal dilation. Single-cell RNA sequencing (scRNA-seq) provides a powerful approach to profile cellular heterogeneity and transcriptional changes during aging; however, generating viable single-cell suspensions from aged LG tissue is technically challenging due to increased fibrosis, cellular fragility, and lipid accumulation. This study presents an optimized, reproducible protocol for the efficient dissociation of aged mouse LGs and the isolation of multiple cell types, suitable for downstream scRNA-seq analysis. Importantly, this protocol is also applicable to young lacrimal glands, conjunctiva, and other exocrine tissues. The workflow employs a stepwise enzymatic dissociation strategy using Collagenase IV, Dispase II, and DNase I for initial tissue digestion, followed by Accutase to dissociate remaining cell clusters and a final DNase I treatment to obtain a clean single-cell suspension. To minimize cell damage, all plasticware is pre-coated with PBS containing bovine serum albumin, and tissue processing is performed under controlled temperature and agitation conditions. Red blood cell lysis and sequential filtration through several strainers further enhance cell purity. Cell type-specific isolation is achieved using reporter mouse lines combined with fluorescence-activated cell sorting (FACS). Using Acta2GFP and PdgfraEGFP mice, we successfully isolated myoepithelial cells and fibroblasts, respectively, following immunostaining with an EpCAM antibody. FACS enabled the separation of the EpCAM⁺/αSMA⁺ myoepithelial population, while EGFP⁺ cells from PdgfraEGFPmice represented the fibroblast population. This protocol yields high-quality, viable single-cell suspensions from aged LGs and can be readily adapted to other fibrotic or aging exocrine tissues requiring gentle dissociation and targeted cell isolation.