Abstract
Phosphoflow is a powerful tool that allows researchers to measure distinct signaling responses to various stimuli in multiple subpopulations of cells. Extension of this technique to mass cytometry (cytometry by time-of-flight or CyTOF) allows many more cell phenotypes and signaling nodes to be interrogated in parallel. The use of fresh whole blood is ideal for capturing the in vivo signaling state of all leukocytes, including granulocytes. In this chapter, we provide a detailed protocol for performing CyTOF phosphoflow in human whole blood, using cytokines and other stimuli. Barcoding and combining of multiple samples and other techniques to reduce batch effects and provide optimal comparability between samples/stimulations are also described.
Key words
- Whole blood
- Phosphoflow
- Mass cytometry
- CyTOF
- Signaling
- Cytokines
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Acknowledgments
The authors thank Rosemary Fernandez for initial protocol development and Prabhu Arunachalam for help in establishing the gating hierarchy. This work was supported by grants 2U19AI057229 and 1U24CA224309 from the National Institutes of Health.
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Sigal, N., Maecker, H.T. (2022). Mass Cytometry Assessment of Cell Phenotypes and Signaling States in Human Whole Blood. In: Barcenilla, H., Diaz, D. (eds) Apoptosis and Cancer. Methods in Molecular Biology, vol 2543. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2553-8_10
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DOI: https://doi.org/10.1007/978-1-0716-2553-8_10
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