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Mouse splenocyte enrichment strategies via negative selection for broadened single-cell transcriptomics
Thomas T Schulze1,2, Andrew J Neville1, Ryan C Chapman1
1Department of Biology, University of Nebraska at Omaha, Omaha, NE 68182, USA.
Insights
This study presents a method to enrich rare immune cells from mouse spleens. The technique improves single-cell transcriptomic analysis by revealing a wider range of cell populations.
Area of Science:
- Immunology
- Cell Biology
- Genomics
Background:
- Mammalian splenic tissue contains numerous immune cells, predominantly lymphocytes.
- High lymphocyte abundance can obscure the detection of low-abundance immune cell populations in analyses.
- Accurate characterization of all immune subsets is crucial for understanding immune responses.
Purpose of the Study:
- To develop a protocol for enriching minority immune cell populations from mouse spleen.
- To enable a more comprehensive analysis of the splenic immune landscape.
- To improve the resolution of single-cell transcriptomic studies.
Main Methods:
- Utilizing immunomagnetic negative depletion to remove abundant cell types.
- Generating an untouched, enriched fraction of rare immune cells.
- Controlled repopulation of enriched cells with untouched splenocytes.
- Validation using flow cytometry and single-cell transcriptomic analysis.
Main Results:
- Successful enrichment of low-abundance immune cell populations.
- Generation of an untouched, viable enriched cell fraction.
- Demonstrated broadened cellular landscape in single-cell transcriptomic clustering analysis.
- Validation of cell integrity and population representation via flow cytometry.
Conclusions:
- The described protocol effectively enriches rare immune cells from mouse spleen.
- This method enhances the cellular landscape revealed by single-cell transcriptomics.
- The protocol provides a valuable tool for detailed immunological studies.
Abstract:
Mammalian splenic tissue is rich in functional immune cells, primarily lymphocytes which can mask low-abundance populations in downstream analyses. This protocol enriches minority immune cell populations from mouse spleen via immunomagnetic negative depletion to generate an untouched enriched cell fraction. Enriched cells are then spiked with untouched splenocytes in a controlled repopulation, validated by flow cytometry and results in a single-cell transcriptomic clustering analysis with a broadened cellular landscape.
