For membrane potential analysis, SkMCs were loaded for 30 min at RT in the dark with a voltage-sensitive dye using the FluoVolt Membrane Potential Kit (Thermo Fisher), following manufacturer instructions, and the fluorescence were evaluated at the cyotofluorimeter (Attune NxT–Thermo Fisher). 20.000 were acquired to set basal conditions and then stimulated with absinthin (10 μM) and acetylcholine (100 μM) alone or combined. Data were expressed and analysed as delta of mean fluorescence intensity (MFI) before and after the stimulus.
Fluovolt membrane potential kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The FluoVolt Membrane Potential Kit is a fluorescence-based assay designed to measure changes in membrane potential in living cells. The kit provides a fluorescent dye and a protocol for monitoring membrane potential dynamics in real-time.
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Lab products found in correlation
Attune nxt, by Thermo Fisher Scientific (2 mentions)
Pluronic f 127, by Thermo Fisher Scientific (2 mentions)
Rc 49mfs, by Warner Instruments (2 mentions)
Tw 2000, by Nikon (2 mentions)
Matlab, by MathWorks (2 mentions)
Cytochalasin d, by Merck Group (2 mentions)
Indo 1 am, by Dojindo Laboratories (1 mentions)
Blebbistatin, by Fujifilm (1 mentions)
Micam02 imaging system, by BrainVision (1 mentions)
Streptomyces conglobatus, by Merck Group (1 mentions)
Cellmask, by Thermo Fisher Scientific (1 mentions)
A23187, by Merck Group (1 mentions)
Blebbistatin, by Abcam (1 mentions)
Di 4 anepps, by Thermo Fisher Scientific (1 mentions)
Rhod 2 am, by Abcam (1 mentions)
D9542, by Merck Group (1 mentions)
Lsrfortessa, by BD (1 mentions)
1 2 dipalmitoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Filter set 46 he, by Zeiss (1 mentions)
16 protocols using fluovolt membrane potential kit
1
Membrane Potential Analysis of SkMCs
2
Membrane Potential Quantification Protocol
3
Fluorescent Membrane Potential Assay
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The FluoVolt membrane potential kit obtained from ThermoFisher Scientific was employed for the experiment. To prepare a fresh FluoVolt loading solution, 12 μL of FluoVolt dye (1000X concentration), 120 μL of 100X PowerLoad concentrate, and 12 mL of PBS were combined in a 15 mL centrifuge tube and thoroughly mixed. For adherent cells, the growth medium was discarded, and the cells were washed twice with PBS. Subsequently, 1 mL of the FluoVolt loading solution was added to each well containing cells, and the cells were incubated at 37 °C for 30 min to allow for dye uptake. Prior to fluorescence measurements, the cells in the FluoVolt loading solution were washed twice with PBS to remove any unbound dye and suspended in PBS. The fluorescence intensity emitted by these cells was then quantified using a fluorescence plate reader, with appropriate wavelength settings of excitation at 522 nm and emission at 535 nm.
4
Cardiac Myocyte Membrane Potential Dynamics
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AP recordings were taken from CMs that had been freshly isolated and from CMs that had been cultured for 24 h. Phenol red can produce fluorescence [28 (link)] potentially generating artefacts in the AP traces so all recordings were performed in reduced serum DMEM/F-12 absent of phenol red. FluoVolt™ membrane potential kit (ThermoFisher Scientific) was used to detect changes in the CM membrane potential. AP measurements were performed at 37 °C within a darkened room. Cells were field stimulated at 1 Hz and using 40 V pulses and allowed to acclimatise for 5 min prior to the first recording. Next, 470 nm light used to excite the FluoVolt™ within the CMs and changes in membrane potential detected by CellOPTIQ software (Clyde Biosciences, UK) over a 10 s period. APs were analysed using the CellOPTIQ software, where APs were averaged and the average TRise (time taken from 10-90 % depolarisation) and APD80 values recorded for each cell.
5
Calcium Transient and Membrane Potential Measurement
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For calcium transient, the cells were loaded with 5 μM Indo-1 AM (1006; Dojindo) dissolved in FluoroBrite Dulbecco’s modified Eagle’s medium (DMEM) (A1896701; Gibco) containing 0.1% pluronic F-127 (P3000MP; Thermo Fisher Scientific) and incubated at 37°C for 1 h. After washing twice with PBS, the cells were incubated in FluoroBrite DMEM for 30 min at 37°C. For measuring membrane potential, the prepared cells were treated with the FluoVolt membrane potential kit (F10488; Thermo Fisher Scientific) for 30 min at 37°C. The medium was replaced with FluoroBrite DMEM containing 2% FBS and incubated for another 30 min. The calcium transient and membrane potential were measured under pacing at 1 Hz. The imaging data were acquired and analyzed using the FDSS/uCELL system (C13299; Hamamatsu Photonics, Hamamatsu, Japan).
6
Optical Action Potential Imaging of hiPSC-CMs
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hiPSC-CMs were loaded with a voltage-sensitive dye (FluoVolt™ Membrane Potential Kit, F10488, ThermoFisher SCIENTIFIC) for 30 min at RT. The excitation and emission wavelengths were 522 and 535 nm, respectively. Then, 10 µM blebbistatin (Wako, 027-17043), an excitation-contraction uncoupler, was applied to avert motion artifacts. All experiments were performed at 37°C under aerial conditions. Optical action potential (OAP) imaging was acquired at a sampling rate of 5 or 10 ms per frame using the MiCAM02 imaging system (Brainvision, Tokyo, Japan) equipped with a high-speed CMOS camera, alongside field-of-view and spatial resolution, which were 5.76 × 4.8 mm and 30 × 30 μm, respectively. OAP parameters including average CL, d (−F)/dtmax, and APD, were calculated using OriginPro 8.6J software (LightStone, Tokyo, Japan).
7
FluoVolt Membrane Potential Assay
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For FluoVolt experiment, 20 μL of packed RBCs were incubated with 1 mL of Hank's Balanced Salt Solution with calcium and magnesium (HBSS+/+) and FluoVolt™ Membrane Potential Kit (Thermo Fisher) was added to the solution. RBCs were incubated at 37°C for 15 min in a rotating block. For generation of labelled RBC‐MVs, CellMask™ (Thermo Fisher) (1:1000 dilution) was added to 100 μL of packed RBCs diluted in 5 mL of HBSS+/+. Cells were incubated at 37°C for 15 min in a rotating block. Then, Ionomycin 10 μM (Streptomyces conglobatus, I9657, Sigma‐Aldrich) or A23187 10 μM (C7522, Sigma‐Aldrich) were added to the cells and incubated at 37°C for 1 h in a rotating block. Cells were centrifuged twice at 2500 × g for 15 min (brakes off), and the supernatant was concentrated to 500 μL using a 10 kDa Amicon® (Millipore Sigma). RBCs‐MVs were purified by loading the concentrate into a qEV 35 nm size‐exclusion chromatography (SEC) column (Izon Science, Christchurch, New Zealand). Twenty 500 μL fractions were collected. For MVs analysis, the fractions 6–9 were pooled together and concentrated to 100 μL using a 100 kDa Amicon filter units.
8
Membrane Potential Measurement Toolkit
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Blebbistatin and Rhod2-AM were purchased from Abcam (Cambridge, UK). The FluoVolt membrane potential kit and di-4 ANEPPS were purchased from ThermoFisher (Waltham MA, US). Latex scattering beads were purchased from Sigma (St Louis MO, US).
9
Evaluating iPSC-derived Cardiomyocyte Membranes
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Patient‐specific iPSC‐derived cardiomyocytes seeded on 35‐mm glass bottom dishes were stained with a FluoVolt Membrane Potential Kit (Thermo Fisher Scientific). FluoVolt images were captured using the same device and the same setting used for Ca2+ imaging.
10
Membrane Potential Measurement in F11 Cells
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F11 cells were seeded at a density of 2,500 cells/well into clear, flat-bottomed, black-walled, 384-well plates pretreated with 30 μg/ml poly-D-lysine. Twenty-four hours later, the culture medium was replaced with differentiation medium. Seventy-two hours later, cells were loaded in HBSS with HEPES (pH = 7.4) supplemented with the dye (1:1,000) and with the PowerLoad Concentrate (1:100), both included in the FluoVolt Membrane Potential Kit (F10488; Thermo Fisher Scientific), for 30 min at 37°C. Then, the background suppressor (Neuro Background Suppressor) diluted 1:20 in HBSS with HEPES was added to the wells. Changes in membrane potential after the addition of KCl were measured with an FDSS7000EX Functional Drug Screen System with a light source at 470–495 nm and emission in the 515–575 nm range. FDSS tips were pretreated with a solution of 0.1% BSA diluted in assay buffer.
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