Viability-Compatible Preservation for Mass Cytometry Using Cisplatin Pulse Quenching and Gradual Formaldehyde Release

ObjectiveMass cytometry (CyTOF) enables high-dimensional single-cell profiling, but especially for non-blood derived samples relies on immediate processing, limiting its application to fragile, low-yield clinical specimens such as urine. We developed a novel preservation protocol that combines viability staining with gentle fixation, enabling cryopreservation and delayed processing. MethodsBuilding on a previously established protocol using imidazolidinyl urea (IU) and MOPS buffer for flow cytometric applications, we enhanced the protocol for CyTOF by incorporating a one-minute pulse of cisplatin (5{square}{micro}M) for live-dead discrimination. A novel quenching step with DL-methionine (5{square}mM) was introduced to minimize background signal without compromising antigen integrity. Samples were fixed overnight at 4{square}{degrees}C and cryopreserved prior to CyTOF analysis. The protocol was validated using peripheral blood mononuclear cells (PBMCs) and was applied to urine samples. ResultsThe preservation method maintained single-cell integrity, surface marker expression, and cisplatin staining specificity. DL-methionine efficiently quenched residual cisplatin reactivity. The protocol performed robustly across a wide range of cell inputs and maintained consistency following freeze-thaw cycles. In urine samples, immune cell subset frequencies and viability discrimination were comparable between the optimized and standard staining protocols. ConclusionOur protocol enables delayed processing and cryopreservation of fragile clinical samples while preserving compatibility with mass cytometry workflows. By integrating cisplatin-based viability staining, DL-methionine quenching, and slow-release fixation, this method supports standardized immune profiling of low-abundance samples in clinical and translational research.