Oxygen-stable streptolysin O variant enables mitochondrial ATP synthesis analysis in minimal cells blood and tissues

Quantifying mitochondrial ATP synthesis remains inaccessible in rare cells, blood, and microdissected tissues because of the biochemical instability of streptolysin O (SLO)-based permeabilization. Here, we introduce an oxygen-stable SLO variant, streptolysin O tolerant (SLOT), and an improved mitochondrial ATP synthesis capacity (iMASC) assay to enable sensitive analysis across diverse samples. SLOT combines non-essential N-terminal deletion (Δ1-77) with a C530A substitution, conferring reductant-independent activity and long-term stability. Temperature-controlled activation restricts permeabilization to the plasma membrane, preserving mitochondrial function. iMASC assay supports continuous measurement for more than 1 h from 10 to 20 cells and applies to adherent and suspension cells, whole blood, and microdissected tissues. Using this platform, we identify platelets as dominant contributors to glycerol-3-phosphate-driven ATP synthesis in murine blood and enable sequential dissection of respiratory chain and ATP synthase activities within single tissues. This framework establishes a broadly applicable approach for mitochondrial metabolism analysis.