The ability to analyze the proteome of single cells is critical for the advancement of studies of steady‐state and pathological processes. Mass cytometry, or CyTOF, combines principles of mass spectrometry and flow cytometry to enable single‐cell analysis of protein expression. CyTOF can simultaneously assess DNA content and proteins and has the capacity to measure 40 to 100 parameters in each cell. Applying this technology to tissues or cells on slides, termed imaging mass cytometry (IMC), allows for visualization of normal and diseased tissues in situ. The high‐dimensional proteomic analysis that can be undertaken with CyTOF and IMC has the potential to enhance our understanding of complex and heterogeneous developmental and disease pathways. This article will describe the CyTOF experimental workflow, including reagent selection, sample preparation, and data analysis. CyTOF is compared to flow cytometry, focusing on the strengths and weaknesses of these powerful techniques. Importantly, we review key studies in mouse models of human disease that highlight the strength of CyTOF and IMC to drive discovery research and therapeutic advancement. © 2021 Wiley Periodicals LLC.

PRDM14 is an epigenetic regulator known for maintaining embryonic stem cell identity and resetting potency in primordial germ cells. However, hematopoietic expression of Prdm14 at supraphysiological levels results in fully penetrant and rapid-onset T-cell acute lymphoblastic leukemia (T-ALL) in the mouse. Here, we show that PRDM14-induced T-ALLs are driven by NOTCH1, a frequently mutated driver of human T-ALL. Notch1 is activated in this murine model via RAG-dependent promoter deletions and subsequent production of truncated, ligand-independent protein from downstream regions of the Notch1 locus. These T-ALLs also have focal changes in H3K4me3 deposition at the Notch1 locus and global increases in both H3K4me1 and H3K4me3. Using a PRDM14-FLAG mouse model, we show that PRDM14 binds within an intron of Notch1 prior to leukemia development. Our data support the idea that PRDM14 binding promotes a chromatin state that allows access of the RAG recombinase complex to cryptic RAG signal sequences embedded at the Notch1 locus. Indeed, breeding into a RAG recombination-deficient background abrogates T-ALL development and prevents Notch1 deletions, while allowing for transient hematopoietic stem cell (HSC)-like pre-leukemia cell expansion. Together, our data suggest that PRDM14 expands a progenitor cell population while promoting a permissive epigenetic state for the creation of driver mutations (here, in Notch1), enabling cancer development through the misappropriation of endogenous cellular DNA recombination machinery.