Multiparametric senescent cell phenotyping reveals targets of senolytic therapy in the aged murine skeleton

Published: 13 July 2023| Version 2 | DOI: 10.17632/hg7sd7hbk5.2
Madison Doolittle,


In the present study we leverage the power of mass cytometry by time-of-flight (CyTOF) to define, at the single-cell level, mesenchymal bone and marrow senescent cells in mice. Specifically, rather than relying principally on p16 or p21 expression, we used multiple criteria to define senescent cells. We assessed senescent cell burden in established skeletal cell populations and tested their susceptibility to senolytic clearance through either genetic or pharmacologic approaches.


Steps to reproduce

Mice were euthanized according to standardized and approved IACUC protocols. Femurs and tibiae were isolated, cleaned of soft tissue, cut at both ends, and marrow centrifuged out of the diaphyses and metaphyses into a collection tube. Marrow was resuspended in 1mg/mL Liberase DL (Sigma) diluted in FACS buffer (0.5% BSA [Sigma] in PBS) and digested at 37°C for 30 minutes to increase yield of stromal cells released from the vasculature fraction as previously described 33. Diaphyses and metaphyses cleared of bone marrow were gently crushed, rinsed in PBS, and then digested in 300 Units/mL of Collagenase IA (Sigma), diluted in MEM α (ThermoFisher), 3 times for 25 minutes each. Bone and marrow solutions were then combined and treated with RBC lysis buffer (ThermoFisher) to clear erythrocytes. The sample was then depleted of cells expressing hematopoietic lineage markers (CD5, CD45R [B220], CD11b, Gr-1 [Ly-6G/C], 7-4, and Ter-119) using Magnet Assisted Cell Sorting (MACS) and the Lineage Cell Depletion Kit (Miltenyl Biotec). Custom conjugated antibodies were generated in-house through the Mayo Clinic Hybridoma Core using Maxpar X8 Ab labeling kits (Fluidigm) according to the manufacturer’s protocol. Isolated cells were resuspended in 1 mL of Cell Staining Buffer (CSB) (Fluidigm) and incubated for 5 minutes with 0.5 µm Cisplatin solution (Fluidigm) in PBS. Samples were then washed twice with CSB. An antibody cocktail of the entire phenotyping panel was prepared as a master mix prior to adding 50 µL of cocktail to samples resuspended in 50 µL of CSB. Samples were then incubated at room temperature for 45 minutes. Samples were washed twice then fixed with 2% PFA (Fluidigm) in PBS. After fixation and wash, samples were resuspended in 30 nM intercalation solution (Fluidigm) and incubated overnight at 4°C. On the following morning, cells were washed with PBS and resuspended in a 1:10 solution of calibration beads and cell acquisition solution (CAS) (Fluidigm) at a concentration of 0.5x106 cells/mL. Prior to data acquisition, samples were filtered through a 35 µm blue cap tube (Falcon). The sample was loaded onto a Helios CyTOF system (Fluidigm) and acquired at a rate of 200-400 events per second. Data were collected as .FCS files using the Cytof software (Version 6.7.1014). After acquisition, intra-file signal drift was normalized to acquired calibration bead signal using Cytof software. Files were uploaded to Cytobank and gated to remove dead cells, debris, and remaining CD45+ cells. Figures in manuscript were generated using either CITRUS or FlowSOM clustering within Cytobank along with dimensionality reduction by either viSNE or UMAP.


Mayo Clinic Rochester


Mass Spectrometry, Mouse, Cellular Senescence, Bone