Flow Cytometry Study of Immune Cell Subpopulations from theMouse Vertebral BoneMarrow and Intervertebral Disc Following Endplate MicrofractureOA

Objective:Although endplate(EP)injury may cause intervertebral disc(IVD)degeneration andModic changes(MCs)in the vertebral bone marrow(VBM),EP injury-induced synchronous cellular reactions and their crosstalk in the IVDandVBMremain unclear.This protocol-based study aimed to streamline and optimize themethods of tissue harvest and cell preparation for flow cytometry(FCM)analysis of T-cell and macrophage subpopulations in both VBM and IVD adjacent to the surgically induced EP microfracture in mice.Methods:EP injury or sham procedure was performed at the spinal levels L4-5 and L5-6 in male mice.Step-by-step techniques on the harvest of lumbar VBMand IVDtissues,isolation of tissue-specific cells,and generation of single cell suspensions were described.FCM analysis was performed using specific antibodies against cell-surface markers and multi-color cytometry for precise delineation of cell subsets to explore the cellular mechanism ofMCs.An extracellular staining assay to identify macrophage subsets,as well as extra-and intra-cellular staining assays to identify T lymphocyte subsets fromthe VBM and IVD,were performed and optimized.Results:FCM analysis demonstrated that significantly increased macrophage numbers and M2a polarization response were observed in VBM samples from the sham and EP injury groups,while the ratio of M2a/total number of macrophages was significantly increased and the ratio of M1/M2a was significantly decreased in IVDs from the sham and EP injury groups.A significantly increased Treg cell response was detected in VBM samples from the EP injury group,but not the sham group.Conclusions:This protocol reports novel and reproducible methods of tissue harvest,cell preparation,and antibody selection for flow cytometry analysis of T-cell and macrophage subpopulations isolated from the VBM and IVD following EP injury inmice.This protocolmay be utilized for exploring the cellular mechanism of MCs and IVD degeneration in animal models.