Measurement of intracellular reactive oxygen species in mature single skeletal muscle fibres by dichlorofluorescein‐based fluorescence microscopy

Abstract

Skeletal muscle fibres continually produce reactive oxygen species (ROS), which modulate several functions in muscle cells. The present study describes the validation of a novel technique to measure intracellular ROS in isolated mature mouse skeletal muscle fibres. Single muscle fibres were isolated from the Flexor Digitorum Brevis muscle of mice, cultured on collagen coated plates, loaded with 17.5 μM 2′, 7′-dichlorodihydrofluorescein-diacetate and measurements in gray scale were taken by fluorescence microscopy every 15 minutes over 45 minutes. Experimental conditions: (i) Addition of 1 μM H2O2 to the extracellular medium induced a 48% increase in intracellular ROS activity. However, this effect was not observed when fibres had been previously treated with 5 mM glutathione ethyl ester (GSHEE). (ii) Contractile activity of isolated fibres, induced by electrical stimulation, caused a moderate increase of 14% in intracellular ROS activity. (iii) Fibres that had been incubated with 5 mM GSHEE for 4 hours did not show any significant increase of intracellular ROS activity after contractile activity. These data confirm that dichlorofluorescein-based fluorescence microscopy is able to detect small changes of intracellular ROS activity. In addition, contractile activity in muscle fibres generated a net increase of intracellular ROS activity, which was abolished when the intracellular antioxidants were enhanced by glutathione loading. This work was supported by The Wellcome Trust. UK.