Cardiomyocytes that adhered to the coverslips were equilibrated in KHB containing 1 mM Ca2+ for 20 min. at 37°C, as previously described 6 (link). The cardiomyocyte suspension was then placed in a Plexiglas chamber, which was positioned on the stage of an inverted epifluorescence microscope (Diaphot 200; Nikon, Tokyo, Japan). Cardiomyocyte contraction was field-stimulated by a Grass S5 stimulator (0.5 Hz, square waves; Grass Technologies, An Astro-Med, Inc., West Warwick, RI, USA), and contractions were videotaped and digitized on a computer. A video edge motion detector (Crescent Electronics, Windsor, ON, Canada) was used to measure cardiomyocyte length and cell shortening, from which the per cent fractional shortening (% FS) and maximal rates of contraction and relaxation (±dl/dt) were calculated 13 (link). All data were analyszed using software from Felix 1.1 software (Photon Technology International, Birmingham, NJ, USA) and IonWizard (IonOptix Corp., Milton, MA, USA).
Grass s5 stimulator
Manufactured by Natus
Sourced in United States
The Grass S5 stimulator is a laboratory instrument used to generate and deliver electrical stimuli to biological samples or subjects. It provides precise control over the timing, intensity, and frequency of the electrical pulses. The S5 is designed for use in a variety of research applications, such as neurophysiology, muscle function studies, and sensory perception experiments.
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4 protocols using grass s5 stimulator
1
Cardiomyocyte Contractility Measurement
2
Measuring Cardiomyocyte Contractility and Ca2+ Signaling
3
Measuring Cardiomyocyte Contraction and Calcium Transients
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Cell shortening and Ca2+ transients were measured at room temperature (22–23°C) in separate experiments. Myocytes were field stimulated to contract by using a Grass S5 stimulator through platinum electrodes placed alongside the bath (0.5 Hz, bipolar pulses with voltages 50% above myocyte voltage threshold). Contractions of myocytes from random fields were videotaped and digitized on a computer. For Ca2+ signal measurements, cells were loaded with 2 μmol/L of Fura‐2/AM (Life Technologies) and alternately excited at 340 and 380 nm by use of a Delta Scan dual‐beam spectrophotofluorometer (Photon Technology International, Edison, NJ) at baseline conditions. Ca2+ transients were expressed as the 340/380‐nm ratios of the resulting 510‐nm emissions. Data were analyzed by the use of Felix software (Photon Technology International).21
4
Sciatic Nerve Stimulation Induces Muscle Contractions
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Animals were anesthetized (2.5% isoflurane) and the sciatic nerve stimulated as described previously.13 Briefly, the sciatic nerve was exposed by surgically opening the fascia between the vastus lateralis and the biceps femoris of the rats. The nerve was hooked onto a platinum electrode, connected to a Grass S5 stimulator (Grass Instruments, USA) and stimulated at a frequency of 100 Hz and 4‐6 V, causing eccentric contractions of the tibialis anterior and EDL muscles.14 Each contraction lasted 2 seconds, with 10 seconds delay before the next contraction. After six repetitions, the animals rested for 1 minute, before the next set was performed. Every animal was stimulated for six sets of six repetitions, a total of 36 contractions. Following the end of the stimulation, the incision site was sutured, and the animals received 0.1 mg/kg buprenorphine (0.03 mg/mL solution; JHP Pharmaceuticals, USA) as analgesic. We collected the tibialis anterior and the EDL muscles 24 hours after conclusion of the stimulation.
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