Measurement of oxygen consumption by murine tissues in vitro

Introduction: A novel in vitro system was developed to measure O₂ consumption by murine tissues over several hours. Methods: Tissue specimens (7-35 mg) excised from male Balb/c mice were immediately immersed in ice-cold Krebs-Henseleit buffer, saturated with 95% O₂:5% CO₂. The specimens were incubated at 37 °C in the buffer, continuously gassed with O₂:CO₂ (95:5). [O₂] was determined as a function of time from the phosphorescence decay rates (1/Τ) of Pd(II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. The values of 1/Τ were linear with [O₂]: 1/Τ=1/Τo+kq [O₂]; 1/7τo=the decay rate for zero O₂, kq=the rate constant in s^μ1τM^τ1. Results: NaCN inhibited O₂ consumption, confirming oxidation occurred in the mitochondrial respiratory chain. The rate of respiration in lung specimens incubated in vitro for 3.912.4 h was 0.24±0.03±M O₂ min^±1 mg^±1 (mean±SD, n=28). The corresponding rate for the liver was 0.27±0.13 (n=11, t<4.7 h), spleen 0.28± 0.07 (n=10, t<5h), kidney 0.34±0.12 (n=7, t<5h) and pancreas 0.35±0.09 (n=10, t<4h). Normal tissue histology at hour 5 was confirmed by light and electron microscopy. There was negligible number of apoptotic cells by caspase 3 staining. Discussion: This approach allows accurate assessment of tissue bioenergetics in vitro.