Standardised dual-flow cytometry workflows for quantitative assessment of mitochondrial superoxide and mitochondrial mass in live cancer cells and primary multiple myeloma cells.

Mitochondrial metabolism plays a critical role in carcinogenesis and cancer progression. Quantitative assessment of mitochondrial function in live cells remains technically challenging because existing biochemical assays lack single-cell resolution, and microscopy-based approaches are limited in throughput and quantitative reproducibility. Here we describe a robust and reproducible standardised dual-flow cytometry protocol for simultaneous quantitative assessment of mitochondrial superoxide production and mitochondrial mass in live cancer cells and primary patient-derived multiple myeloma plasma cells using MitoSOX Green and MitoTracker Red. The protocol provides a step-by-step workflow comprising preparation of cultured cancer cells or isolation of primary CD138+ plasma cells, optimised probe staining, viability discrimination, standardised flow cytometry acquisition and gating, and quantitative fluorescence normalisation. Compared with conventional mitochondrial assays requiring cell lysis or imaging-based analysis, this approach enables high-throughput, quantitative mitochondrial profiling at single-cell resolution in heterogeneous populations while preserving cellular integrity. The procedure incorporates defined staining conditions, instrument calibration guidance, quality-control criteria and normalisation strategies to improve reproducibility across experiments and laboratories. The workflow yields robust fluorescence measurements with low technical variability and enables discrimination of mitochondrial oxidative activity relative to mitochondrial content, facilitating analysis of mitochondrial dysfunction, oxidative stress responses and treatment-induced mitochondrial perturbations. The method is compatible with multiparametric flow cytometry and can be adapted to diverse cell types and experimental systems. The complete protocol requires ∼6-8 h for cultured cells or 8-12 h when primary cell isolation is included and can be implemented by researchers with standard cell culture and flow cytometry expertise.