Abstract
Cytometry by time-of-flight (CyTOF) enables comprehensive immune profiling for translational research. However, challenges such as signal variability, nonspecific binding, and antibody incompatibility can compromise data quality. This study presents an optimized CyTOF staining protocol for human peripheral blood mononuclear cells and bone marrow aspiration concentrate samples, addressing these challenges by refining antibody conjugation with polymer X8, saponin use, and fixation protocols. Preliminary data indicate improved staining for key markers (CD14, CD16, and CD19), enhancing signal consistency and clarity. These findings advance the utility of CyTOF in orthopaedic research and immune profiling for diseases such as osteonecrosis of the femoral head.
Impact Statement
This study provides a practical and reproducible solution to several persistent technical challenges in CyTOF-based immune profiling of PBMC and BMAC samples. By optimizing conjugation procedures, permeabilization conditions, and fixation strategies, we enhance the reliability of immune phenotyping workflows. These improvements not only support more accurate data interpretation but also pave the way for applying CyTOF in clinical studies on musculoskeletal disorders, offering potential to uncover novel biomarkers and therapeutic targets in ONFH and beyond.
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