Profiling of Natural Killer Interactions With Cancer Cells Using Mass Cytometry.

We developed a comprehensive method for functional assessment of the changes in immune populations and killing activity of peripheral blood mononuclear cells after cocultures with cancer cells using mass cytometry. In this study, a 43-marker mass cytometry panel was applied to a coculture of immune cells from healthy donors' peripheral blood mononuclear cells with diverse cancer cell lines. DNA content combined with classical CD45 surface staining was used as gating parameters for cocultures of immune cells (CD45high/DNAlow) with hematological (CD45low/DNAhigh) and solid cancer cell lines (CD45neg/DNAhigh). This strategy allows for universal discrimination of cancer cells from immune populations without the need for a specific cancer cell marker and simultaneous assessment of phenotypical changes in both populations. The use of mass cytometry allows for simultaneous detection of changes in natural killer, natural killer T cell, and T cell phenotypes and degranulation of immune populations upon target recognition, analysis of target cells for cytotoxic protein granzyme B content, and cancer cell death. These findings have broad applicability in research and clinical settings with the aim to phenotype and assess functional changes following not only NK-cancer cell interactions but also the effect of those interactions on other immune populations.

Simplified mass cytometry protocol for in-plate staining, barcoding, and cryopreservation of human PBMC samples in clinical trials.

With the increasing use of mass cytometry in clinical research, a simplified and standardized protocol for immunophenotyping human peripheral blood mononuclear cells (PBMCs) in clinical trials is needed. We present a simplified in-plate staining protocol for up to 80 samples, for laboratories of all mass cytometry expertise levels, aimed to generate reproducible datasets for large clinical cohorts. In this protocol, we provide details on the requirements to obtain meaningful results, spanning from sample quality, barcoding, and batch-freezing of stained samples.

Mass cytometry staining for human bone marrow clinical samples.

This protocol details a staining technique optimized for immunophenotyping of human bone marrow immune populations using mass cytometry. The protocol accounts for the limitations of working with human bone marrow, such as reduced viability, low cell counts, and fragile cell pellets, to successfully acquire single viable cells ready for downstream analysis. This assay can be used to characterize the activation, exhaustion, and cytotoxicity of immune populations and ensure comprehensive immunophenotyping of human bone marrow clinical samples.