Masterflex c l peristaltic pump

Glycerinated rabbit psoas fibers were dissected into bundles, ranging from 0.3 to 0.5 mm in diameter and 3–5 cm in length and were tied at each end using surgical silk. The tied fiber bundles were then held in place at a fixed length within a 25-µl glass capillary (Drummond Scientific), with the ends of the sutures affixed to the capillary using short sections of silicone tubing. Buffer exchange was accomplished with a Masterflex C/L peristaltic pump (Cole-Parmer) at the flow rate of 0.5 ml/min. Spin-labeled RLC constructs were exchanged onto permeabilized fibers as described previously (Mello and Thomas, 2012 (link)), except that the concentration of DTT in the wash before data acquisition was 0.5 mM instead of 30 mM. TnC was reconstituted during a 1-h incubation at 4°C. Rabbit skeletal TnC was purchased from Life Diagnostics. It was shown previously that this exchange and reconstitution protocol is complete (>90% reconstitution for both RLC and TnC) and has no significant effect on function, as measured by Ca-dependent myofibrillar MgATPase assays (Mello and Thomas, 2012 (link)). Rigor and relaxation solutions used during acquisition were as described previously (Fusi et al., 2015 (link)). Ionic strength and pH during acquisition were maintained at 150 mM and 7.1, respectively.