In Vivo Sortase A-Mediated LPETG Ligation for Rare Cell Enrichment.

Purification of rare cells from tissues typically requires prolonged fluorescence-activated cell sorting (FACS), which may adversely affect cell state. Magnetic-activated cell sorting (MACS) prior to FACS can significantly reduce sorting time; however, a specific surface marker for MACS is not always available for rare or poorly characterized populations. To address this challenge, uLIPSTIC mice, originally designed for studying cell-cell interactions via Cre-inducible SrtA expression, were repurposed for cell enrichment. By administering biotin-conjugated LPETG-SrtA substrate in vivo, efficient labeling of SrtA-expressing cells with anti-biotin microbeads was achieved for subsequent MACS enrichment. This SrtA-based rare cell enrichment (SRACE), when combined with genetic labeling of fluorescent proteins, enables further purification of rare cells by FACS. The effectiveness of SRACE was validated in multiple biological contexts, including lung pan-macrophages, lung perivascular macrophages, and splenic CD11c+ cells. Collectively, SRACE offers a versatile strategy for enriching cell populations, particularly when specific surface markers are lacking, and offers the added advantage of simultaneously capturing donor cells and their interacting partners when such analysis is desired.