Protocol for isolation and cryopreservation of human peripheral blood mononuclear cells for high-resolution respirometry

High-resolution respirometry (HRR) of peripheral blood mononuclear cells (PBMCs) relies on controlled preanalytical procedures. Here, we present an optimized workflow for PBMC isolation from human whole blood, followed by short-term cryopreservation at −80°C. The protocol includes rapid thawing to preserve cell viability, washing steps to minimize platelet contamination, and quality-control assessment of PBMC number, viability, and composition before HRR measurements. Critical handling steps are optimized and validated by HRR against freshly prepared cells.