The dynamic calcium responses to twitch stimuli were performed as previously described [8 (link),44 (link)]. Briefly, lumbrical muscles were incubated with the cell permeant acetoxymethyl (AM) ester form of the low-affinity calcium indicator mag-fura-2 (10 μM) (Catalog #M1292, Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at room temperature. The mag-fura-2 signal was excited by alternating between 344 nm and 375 nm and the emission signal was captured at 510 nm. Pre-loading backgrounds were subtracted from all signals, and the mag-fura-2 fluorescence ratio (344 nm/375 nm) was taken as an undistorted representation of the rapid intracellular calcium transient associated with a twitch contraction.
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