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.