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Published on: June 19, 2019
Optimization of Cytometry by Time-of-Flight Staining for Peripheral Blood and Bone Marrow Samples
Yosuke Susuki1, Issei Shinohara1, Masatoshi Murayama1
1Department of Orthopaedic Surgery, Stanford University School of Medicine, Stanford, California, USA.
Insights
This study optimizes Cytometry by time-of-flight (CyTOF) staining for immune profiling. The refined protocol improves signal quality for key markers in blood and bone marrow samples, advancing translational research.
Area of Science:
- Immunology
- Biotechnology
- Translational Research
Background:
- Cytometry by time-of-flight (CyTOF) is crucial for immune profiling in translational research.
- Existing CyTOF protocols face challenges like signal variability, nonspecific binding, and antibody incompatibility, impacting data quality.
Purpose of the Study:
- To present an optimized CyTOF staining protocol for human peripheral blood mononuclear cells and bone marrow samples.
- To address common challenges in CyTOF data acquisition and improve signal consistency and clarity.
Main Methods:
- Refined antibody conjugation using polymer X8.
- Optimized saponin use and fixation protocols for sample preparation.
- Application to human peripheral blood mononuclear cells and bone marrow aspiration concentrate.
Main Results:
- Preliminary data show improved staining for key immune markers, including CD14, CD16, and CD19.
- Enhanced signal consistency and clarity were observed in the processed samples.
- The optimized protocol addresses challenges of signal variability and nonspecific binding.
Conclusions:
- The optimized CyTOF staining protocol enhances data quality for immune profiling.
- This advancement is particularly relevant for orthopaedic research and understanding immune responses in diseases like osteonecrosis of the femoral head.
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.
