Aged murine bone marrow myeloid and mesenchymal cells develop unique senescence phenotypes
Description
In the present study we leverage the power of mass cytometry by time-of-flight (CyTOF) to define, at the single-cell level, senescence phenotypes of various cell lineages within the murine bone microenvironment. Six folders exist for the main CyTOF experiments that were performed (additional info in the Inventory file): 1) B6_24m_BM: Initial senescence phenotyping of bone marrow immune cells isolated from 24-month-old C57BL/6N mice 2) LysMLOX_24hr_BM: Analysis of LysM-LOX-ATTAC transgene effect in bone marrow immune cells isolated from 24-month-old mice at 24hrs post-treatment 3) LysMLOX_2wk_BM: Analysis of LysM-LOX-ATTAC transgene effect in bone marrow immune cells isolated from 24-month-old mice after 2-weeks of treatment 4) DMPLOX_2wk_Mes: Analysis of DMP1-LOX-ATTAC transgene effect in bone and marrow mesenchymal (CD45-Lin-) cells isolated from 24-month-old mice after 2-weeks of treatment 5) INKATTAC_2wk_BM: Analysis of INK-ATTAC transgene effect in bone marrow immune cells isolated from 24-month-old mice after 2-weeks of treatment 6) B6_Mes_v_Immune: Comparison of senescence phenotypes between bone/marrow mesenchymal (CD45-Lin-) and immune cells in 24-month-old wild-type C57BL/6N mice
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Steps to reproduce
Cleanup of cell debris–including removal of beads, dead cells, doublets (and CD45+ cells in mesenchymal samples)–was performed using Cytobank software. Visual representation of single-cell data was achieved using viSNE mapping (5,000 iterations, 100 perplexity, 0.5 theta), which is based on the t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm. FlowSOM clustering was performed within Cytobank (hierarchical consensus, 10 iterations) and cluster labels were assigned using established literature on immune and mesenchymal bone/marrow cell types, with relative marker intensities per cluster visualized by heatmap. Quantified values were exported to Graphpad Prism 10 to construct plots and perform statistical analyses.
Institutions
- Mayo Clinic MinnesotaMN, Rochester
- UConn HealthCT, Farmington