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In Vivo Sortase A-Mediated LPETG Ligation for Rare Cell Enrichment
Han-Ying Huang1, Jing-Min Zeng1, Lin Tian2
1State Key Laboratory of Oncology in South China, Guangdong Provincial Clinical Research Center for Cancer, Sun Yat-sen University Cancer Center.
Abstract:
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.
Insights
A novel SrtA-based rare cell enrichment (SRACE) method uses in vivo biotinylation for magnetic-activated cell sorting (MACS) enrichment. This technique effectively isolates rare cells when specific surface markers are unavailable, improving cell purification.
Area of Science:
- Cell biology
- Immunology
- Biotechnology
Background:
- Purifying rare cells often requires lengthy fluorescence-activated cell sorting (FACS), potentially altering cell states.
- Magnetic-activated cell sorting (MACS) can reduce FACS time but lacks specific markers for rare or uncharacterized cell populations.
Purpose of the Study:
- To develop a novel cell enrichment strategy for rare populations lacking specific surface markers.
- To repurpose uLIPSTIC mice for efficient in vivo cell labeling and subsequent enrichment.
Main Methods:
- Administered biotin-conjugated LPETG-SrtA substrate in vivo to label SrtA-expressing cells.
- Utilized anti-biotin microbeads for magnetic-activated cell sorting (MACS) enrichment.
- Combined SrtA-based rare cell enrichment (SRACE) with fluorescent protein labeling for FACS purification.
Main Results:
- Successfully enriched SrtA-expressing cells using in vivo biotinylation and MACS.
- Validated SRACE in diverse cell types, including lung macrophages and splenic CD11c+ cells.
- Demonstrated SRACE's efficacy in purifying rare cells lacking specific surface markers.
Conclusions:
- SRACE provides a versatile method for enriching rare cell populations, especially when surface markers are absent.
- The technique allows for simultaneous capture of interacting cells, aiding in cell-cell interaction studies.
- SRACE enhances cell purification efficiency and preserves cell state compared to prolonged FACS alone.
