Percent SL was recorded with an IonOptix iCCD camera and calculated as follows: ([resting SL−peak SL]×100/resting SL). [Ca2+]i was measured using a dual-excitation spectrofluorometer (IonOptix LLC). The “in vivo” calibration was performed by using solutions containing 10 μmol/L ionomycin (Sigma), and [Ca2+]i was calculated as described previously.29 (link) [Ca+2]i transient (Δ[Ca+2]i) amplitude was considered as: peak [Ca+2]i−resting [Ca+2]i. ΔCa2+ decay parameters and sarcomere relaxation (τ and time to 90% decline) were analyzed by using IonWizard 6.0 software (IonOptix LLC). All resulting data were plotted and further analyzed with Prism 6 software (GraphPad Software, Inc). After Ca2+ reuptake and SL shortening were assessed under steady-state conditions, cardiomyocytes field-stimulated at 4 Hz were treated with increasing doses of ISO (Sigma-Aldrich Co). Thus, [Ca2+]i and SL were studied by superfusing 10−9, 10−8, 10−7, or 10−6 mol/L ISO. The raw data were calibrated and analyzed by using IonWizard 6.0 (IonOptix LLC), and a dose-response curve was then plotted by using Prism 6 software (GraphPad Software, Inc).
Ionwizard 6
Manufactured by IonOptix
Sourced in United States
IonWizard 6.6 is a software application designed for data acquisition and analysis for cellular physiology experimentation. It provides a comprehensive suite of tools for recording and analyzing various ion-related signals, including calcium, sodium, and membrane potential.
Automatically generated - may contain errors
Lab products found in correlation
Fura 2 am, by Thermo Fisher Scientific (4 mentions)
Myocyte calcium and contractility system, by IonOptix (2 mentions)
Myopacer, by IonOptix (2 mentions)
Hyperswitch myocyte calcium and contractility system, by IonOptix (2 mentions)
Myopacer field stimulator, by IonOptix (2 mentions)
Fura 2 am, by Merck Group (2 mentions)
Eclipse ts100, by Nikon (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Leica dm irbe microscope, by Leica Microsystems (1 mentions)
Pmt 300, by IonOptix (1 mentions)
Dual excitation spectrofluorometer, by IonOptix (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Prism 6, by GraphPad (1 mentions)
Fura 2, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Laminin, by Roche (1 mentions)
Field stimulator, by IonOptix (1 mentions)
Fura 2 acetoxymethyl ester, by Thermo Fisher Scientific (1 mentions)
Video based edge detection system, by IonOptix (1 mentions)
Myocam s camera, by IonOptix (1 mentions)
16 protocols using ionwizard 6
1
Cardiomyocyte Calcium Handling Analysis
2
Cardiomyocyte Calcium Transient Analysis
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Calcium transients from spontaneous or electrically evoked activity in neoCM were obtained with the Myocyte Calcium and Contractility System (IonOptix, Westwood, Massachusetts, USA). NeoCM were measured 6–9 days after isolation. NeoCM monolayers were transiently transfected with miR mimics for 24 h as described above. Cells were loaded with 4 µM Fura-2 AM (stock solution: 1 mM in DMSO) at 37°C for 30 min. Each sample was measured three times at room temperature: after recording control signals for 2 min the buffer was changed to 10 µM isoprenaline (ISO) (2 min break) and response to ISO was measured for 3 min and after a 1 min break again for 3 min. If possible, spontaneous calcium transients were recorded. Otherwise, cells were paced at 1 Hz to evoke calcium transients (MyoPacer, 5–10 V per pulse, pulse duration maximum 4 ms). IonWizard 6.6 (IonOptix) was used for data acquisition at sampling frequencies of 100 Hz (average 4: 4 collected data points are averaged into one raw data point) or 250 Hz (average 1). Monotonic transient analysis was performed using IonWizard 6.6.
3
Intracellular Calcium Transient Measurement
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The intracellular Ca2+ ([Ca2+]i) transient was measured as previous described.50 The [Ca2+]i transient was recorded from infected adult cardiac myocytes loaded with 2 μmol L−1 Fura‐2‐AM in the dark at room temperature for 30 minutes. After the loading of Fura‐2, the cells were washed twice with the superfusion buffer containing 1.8 mmol L−1 CaCl2 and kept in the same buffer in the dark at room temperature for 30 minutes. To evoke [Ca2+]i transients, the cells were stimulated at 1 Hz by field stimulation applied by two parallel platinum electrodes. For the determination of the SR calcium content, the cells were treated with 10 mM caffeine. Cytosolic free Ca2+ was measured as the 340 to 380 nm (340/380) ratio of Fura‐2 fluorescence excited at 510 nm using an IonOptix photometry system (PMT‐300; IonOptix, Westwood, MA, USA). Data were acquired and analysed with Signal Averager software IonWizard 6.6 (IonOptix).
4
Optical Assessment of Cardiomyocyte Shortening
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Unloaded CM shortening was assessed optically, as described previously [14 (link)]. Briefly, contractions were triggered by field stimulation, as described above, at room temperature. CM were imaged using an inverted microscope with a 40× air objective using phase contrast (Leica DM IRBE microscope; Leica Microsystems, Vienna, Austria). Sarcomere shortening dynamics were quantified using the IonWizard 6.6 software package (IonOptix, Westwood, MA, USA). CM with a resting sarcomere length (SL) shorter than 1.65 µm were excluded from the analyses.
5
Measuring Cardiomyocyte Calcium Dynamics
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Infected adult cardiomyocytes were incubated with 2 µmol/L fura-2 for 30 min at 37 ℃, and then were washed twice with Tyrodes solution containing 1.8 mmol/L CaCl 2 . We stimulated cells at 1.0 Hz using eld stimulation to evoke Ca 2+ transient. Levels of intracellular Ca 2+ were determined by calculation of the 340 to 380 nm (340/380) ratio of Fura-2 uorescence excited at 510 nm using an IonOptix photometry system (PMT-300, USA). The SR based calcium content in cells was determined by using 10 mM caffeine. Data were collected and analyzed by using SignalAverager Software IonWizard 6.6 (IonOptix) and with all parameters set to manufacture defaults.
6
Cardiomyocyte Viability and Contractility Evaluation
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Cardiomyocyte viability and contractility were evaluated at three time points throughout the experimental protocol: 7 hours after cell isolation, after transfection with siRNA (before ischemia), and at the end of reperfusion (Figure 1 ). The viability of cardiomyocytes was assessed by the trypan blue exclusion test [29 (link)–31 (link)]. Cardiomyocyte contractility was measured using the IonOptix system and IonWizard 6.0 software (IonOptix, Milton, MA USA). After a stabilization period the chamber containing the cells was perfused with an oxygenated buffer at a constant temperature of 37°C. Cells were continuously paced (stimulated) at 1 Hz and 5 V (IonOptix MyoPacer, Milton, MA, USA). The assessment of cardiomyocyte contractility was made by the measurement of peak shortening, maximal velocity of cell shortening, and maximal velocity of cell relengthening [21 (link)] on 8–10 cardiomyocytes per independent experiment, over a 10 min period to give an average measure per sample.
7
Cardiac Calcium Dynamics and Contractility Assay
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Sarcometic contractions and calcium transients were measured as described previously [41 (link)]. The cardiomyocytes from the left ventricle (n = 16–23 in each group) were incubated with 2-μmol/L-Fura-2-AM Tyrode solution for 20 min and then washed with Tyrode solution repeatedly. The sarcomeric contractions and calcium transients were recorded in the Tyrode solution (control), 0.1-μmol/L-NPW Tyrode solution, and 0.1-μmol/L-NPB solution at 37 °C using the Ionoptix HyperSwitch Myocyte Calcium and Contractility System (IonOptix LLC, Westwood, CA, USA). The composition of the Tyrode solution was as follows (in mmol/L): NaCl 137, KCl 4.5, MgCl2 1, CaCl2 2, glucose 10, HEPES 5, and pH adjusted to 7.4 with NaOH. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). During the measurement, the cells were stimulated with a field stimulator (MyoPacer Field Stimulator, IonOptix LLC, Westwood, CA, USA) at a frequency of 1 Hz. The offline analysis was performed with IonWizard 6.5 software (IonOptix LLC, Westwood, CA, USA).
8
Contractility and Calcium Dynamics in CMCs
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In order to evaluate functional properties of CMCs cellular contractility and calcium transients were analyzed. For the experiment CMCs were transferred from cultivation media to normal calcium (2 mmol/l) Tyrode solution. CMCs sarcomeric contractions and calcium transients were measured with Ionoptix HyperSwitch Myocyte Calcium and Contractility System (IonOptix LLC, Westwood, USA), with the Sarclen sarcomere length acquisition module. Cells were loaded with Fura-2 (Molecular Probes, Invitrogen, USA). For stock solution Fura-2-am powder was dissolved in DMSO (Sigma-Aldrich, USA) to reach final concentration of 1 mmol/l. Cells were incubated for 20 min in normal calcium Tyrode solution with 2 μmol/l Fura-2-am and then repeatedly washed with normal calcium Tyrode solution. After 20 min of incubation, measurements followed. Measurements were performed in normal Tyrode solution at 37±0.5 °C. Cells were stimulated with field stimulator (MyoPacer Field Stimulator, IonOptix LLC, Westwood, USA) at 1 Hz.
For offline analysis of sarcomeric contractions and calcium transients the IonWizard 6.5 software (IonOptix LLC, Westwood, USA) was used.
For offline analysis of sarcomeric contractions and calcium transients the IonWizard 6.5 software (IonOptix LLC, Westwood, USA) was used.
9
Intracellular Calcium Dynamics in EDL Muscle
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Intracellular [Ca2+] responses were measured as reported [20 (link)]. An EDL muscle was incubated in either standard PSS or in Ca2+-free PSS containing 3 mM (ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA)). Each solution contained 1 μM Fura 2-AM (Cat. # F4185, Fisher); a muscle was incubated for 60 min at 37 °C and then washed for 20 min to remove excess dye. The 1.2% BaCl2 was then added while fluorescence was recorded at 510 nm during alternative excitation (10 Hz) at 340 nm and 380 nm using a × 20 objective (Nikon Fluor20, numerical aperture (NA) = 0.45). Data were acquired using IonWizard 6.3 software (IonOptix; Milford, MA) on a personal computer and expressed as fluorescence (F) ratios (F340/F380) after subtracting autofluorescence recorded prior to dye loading.
10
Measurement of Ca2+ Transients in NRCs
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The measurement of the fluorometric Ca2+ transients in NRCs was performed as described by Kirschmer et al. (16 (link)). Briefly, cells were cultured on cover slides coated with laminin (Roche) and loaded for 20 min at room temperature with Fura-2 (2 mM) in a Ca2+-free normal Tyrode solution (140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 5 mM Hepes, 10 mM glucose, pH 7.4). Ca2+ transients were measured in normal Tyrode solution supplemented with 1 mM CaCl2 at a pacing frequency of 1 Hz using the “Myocyte and Contractility System” from Ionoptix. Data were corrected for background fluorescence 340/380 and analyzed using the IonWizard 6.3 software (Ionoptix).
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