After preparing the 100 μmol/L di‐8‐ANEPPS in 20%(w/v) Pluronic‐F127 (Invitrogen) in DMSO from a stock solution of 2 mmol/L di‐8‐ANEPPS in DMSO, cells were incubated with lipophilic fluorescent indicator di‐8‐ANEPPS (10 μmol/L, Invitrogen) prepared in the cell culture medium for 15 minutes at 37°C, and then washed thrice using PBS. Cells were then fixed with 4% PFA for 20 minutes at RT, washed again, mounted with Fluoroshield Mounting Medium with DAPI and imaged using the confocal microscope.
Di 8 anepps
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany
Di-8-ANEPPS is a fluorescent probe used in various research applications. It functions as a voltage-sensitive dye, capable of detecting and reporting changes in membrane potential. The core purpose of Di-8-ANEPPS is to provide a tool for researchers to study and analyze electrical activity in biological systems.
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Lab products found in correlation
Pluronic f 127, by Thermo Fisher Scientific (6 mentions)
Fluo 4 am, by Thermo Fisher Scientific (5 mentions)
Lsm 710, by Zeiss (5 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Forskolin, by Cell Signaling Technology (2 mentions)
Blebbistatin, by Merck Group (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Di 8 anepps, by Olympus (2 mentions)
Phenol red free dmem, by Merck Group (1 mentions)
Sp5 x mp inverted laser scanning confocal microscope, by Leica (1 mentions)
Forskolin, by Thermo Fisher Scientific (1 mentions)
Cellbrite orange cytoplasmic membrane dye, by Biotium (1 mentions)
Concanavalin a alexa fluor 647 conjugate, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Cellmask orange, by Thermo Fisher Scientific (1 mentions)
Fitc labeled goat anti rabbit igg h l, by Beyotime (1 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
Mouse laminin, by BD (1 mentions)
Hoechst 33342, by Bio-Techne (1 mentions)
Vt1000s vibratome, by Leica (1 mentions)
52 protocols using di 8 anepps
1
Staining Cells with Di-8-ANEPPS
2
Cardiomyocyte Imaging with Fluorescent Dyes
3
Visualizing Cardiomyocyte Tubular Network and Calcium Dynamics
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When studied alone, the tubular network of isolated cardiomyocytes was visualised in CM stained with 10 µM di-8-ANEPPS (Molecular Probes) with a confocal microscope (Olympus FV1000) equipped with an x60 oil immersion objective. Dye was excited at 488 nm and fluorescence collected between 505 and 605 nm.
For simultaneous calcium transients and tubular network studies, Fluo-4 AM (5 µM) and di-8-ANEPPS (Molecular Probes) double-labelled cells were used. To record calcium transients, CM were scanned with confocal microscopy (Olympus FV1000) at 500 Hz along a line perpendicular to their longitudinal axis avoiding the nuclear area. Fluo-4 and di-8-ANEPPS dyes were both excited at 488 nm and fluorescence intensities were collected between 505 nm and 525 nm and beyond 660 nm, respectively. During the acquisition, CM were electrically field stimulated with square pulses (1 Hz, 2 ms) and superfused at 32 °C with Tyrode’s solution. To record 2D calcium transients (XYT), the same experimental conditions were used except that the confocal microscope was a Dynascope Zeiss LSM710 NLO. Each video of calcium transients was acquired with a frame rate between 550 and 270 Hz depending on the cell size and orientation. Frame sizes were between 512 × 100 pixels and 512 × 200 pixels. Pixel size was 310 nm.
For simultaneous calcium transients and tubular network studies, Fluo-4 AM (5 µM) and di-8-ANEPPS (Molecular Probes) double-labelled cells were used. To record calcium transients, CM were scanned with confocal microscopy (Olympus FV1000) at 500 Hz along a line perpendicular to their longitudinal axis avoiding the nuclear area. Fluo-4 and di-8-ANEPPS dyes were both excited at 488 nm and fluorescence intensities were collected between 505 nm and 525 nm and beyond 660 nm, respectively. During the acquisition, CM were electrically field stimulated with square pulses (1 Hz, 2 ms) and superfused at 32 °C with Tyrode’s solution. To record 2D calcium transients (XYT), the same experimental conditions were used except that the confocal microscope was a Dynascope Zeiss LSM710 NLO. Each video of calcium transients was acquired with a frame rate between 550 and 270 Hz depending on the cell size and orientation. Frame sizes were between 512 × 100 pixels and 512 × 200 pixels. Pixel size was 310 nm.
4
Vesicle Flow Cytometry of Extracellular Vesicles
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Isolated EVs were subjected to vesicle flow cytometry (vFC) using a specific fluorescent membrane dye Di8-ANEPPS (D3167, Thermo-Fisher) as previously described30. Briefly, EV samples were labeled with Di8-ANEPPS staining buffer (12 μM) supplemented with Pluronic F-127(#P6866, Thermo-Fisher) at room temperature for 30 min and then stained with following APC-conjugated antibodies listed in Table 1 . Configuration of the MACSQuant10 (MQ10) was set to trigger on events over 1.1 V on the basis of Di8-ANEPPS (B3 channel on the MQ10 to focus analysis on dye-labeled events. Additional details are provided in the Supplementary information . To identify detergent soluble EVs and to avoid ‘swarm’ and other background artifacts, samples were analyzed twice, first without detergent and then second after the additive of the detergent Triton X-100 (T8532, Sigma Aldrich, St. Louis, MO, USA). This sequential strategy enables the selection of a gate that identifies detergent soluble Di8-ANEPPS positive EVs from which additional staining with high index fluorophore such as APC can be used for the detection of antibodies bound to EVs.
5
Membrane dipole potential assay
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RN cultures were seeded at 4000 cells/well in a 96 well plate and permitted to settle for 24 h before washing well before labelling with 0.5 μM of the fluorescent probe di-8-ANEPPs (Invitrogen, from 2 mM stock solution in ethanol) for 1.5 h in phenol-red free DMEM (Sigma-Aldrich) (Davis et al., 2015 ). After this time the ratiometric di-8-ANEPS fluorescence intensity at excitation of 420/520 nm and emission of 670 nm using a Safire plate reader for each cell population was recorded before and 10 min after cells were treated with varying concentrations of TPGS for 10 min. The change in fluorescence ratio of di-8-ANEPPs indicates a change in the membrane dipole potential on addition of an agent of interest. The dissociation constant (Kd) of the interaction of TPGS for neuronal cells was determined by fitting the change in di-8-ANEPPs fluorescence ratio to a hyperbolic binding equation as described previously (Davis et al., 2010 (link)).
6
Simultaneous Calcium and Membrane Potential Imaging
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Cytoplasmic Ca2+ concentration was monitored, loading the cells with Fluo-4-AM (4 μg/ml, Invitrogen) for 20 min. Cells were perfused at 37 °C with normal Tyrode solution (140 mM NaCl, 4.5 mM KCl, 10 mM glucose, 10 mM HEPES, 1 mM MgCl2, 1.8 mM CaCl2; pH 7.4) and field stimulated at 1 Hz (30 V, 10 ms). A 470 nm LED was used to excite the fluorophore, with emission passing through a 560±35 nm filter, before being recorded by a NeuroCMOS camera (0.25 kHz, Redshirt, Cairn Research, Kent, UK).
Membrane potential was monitored by loading di-8-ANEPPs (5 μM, Invitrogen) for 10 min. The excitation-contraction uncoupler, blebbistatin (1 μM, Sigma), was used to minimise motion artefact. The conditions used above were used, with a 535 nm LED and 590 nm long-pass filter, recording at 1 kHz.
Calcium and action potential data were analysed using pClamp10 (Axon CNS Molecular Devices, Sunnyvale, CA, USA).
Membrane potential was monitored by loading di-8-ANEPPs (5 μM, Invitrogen) for 10 min. The excitation-contraction uncoupler, blebbistatin (1 μM, Sigma), was used to minimise motion artefact. The conditions used above were used, with a 535 nm LED and 590 nm long-pass filter, recording at 1 kHz.
Calcium and action potential data were analysed using pClamp10 (Axon CNS Molecular Devices, Sunnyvale, CA, USA).
7
Measuring Cardiomyocyte Morphology Using Di-8-ANEPPS
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Cardiac tissues were incubated with 10 μM di-8-ANEPPS (Invitrogen, Carlsbad, CA) for 15 min at 37°C to stain the cell membrane. Tissues were then rinsed with normal Tyrode’s solution (1.8 mM CaCl2, 5 mM glucose, 5 mM HEPES, 1 mM MgCl2, 5.4 mM KCl, 135 mM NaCl, 0.33 mM NaH2PO4, pH 7.4) (Sigma Aldrich, St. Louis, MO) and imaged at 37°C using a Leica SP5 X MP Inverted Laser Scanning Confocal Microscope (Leica, Wetzlar, Germany) with a 63x glycerin objective. The cell borders were traced manually in ImageJ (NIH, Bethesda, MD), the cell area was then measured by fitting the traced area to an ellipse, and the aspect ratio was determined using the ratio of the major axis (length of cell) to the minor axis (width of cell).
8
BeWo cell fusion induced by forskolin
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BeWo cells were incubated with 20 μM forskolin (3828, Cell Signaling Technology) to induce cell fusion. After 48 h, the cultured cells were stained with Hoechst 33342 (H1399, Invitrogen) and 2 μM Di-8-ANEPPS (D3167, Invitrogen) for 30 min, and subsequently imaged by Zeiss LSM 880 Fast Airyscan Confocal system (Carl Zeiss, Jena, Germany).
In fluorescent images of six randomly selected fields from three or more independent experiments, the number of nuclei within non-fused or fused cells was counted. Cells with more than two nuclei were identified as fused cells, and the fusion index in each group was calculated as follows: (the number of nuclei in fused cells) / (total number of nuclei). The fusion index data were expressed relative to the forskolin-untreated WT control.
In fluorescent images of six randomly selected fields from three or more independent experiments, the number of nuclei within non-fused or fused cells was counted. Cells with more than two nuclei were identified as fused cells, and the fusion index in each group was calculated as follows: (the number of nuclei in fused cells) / (total number of nuclei). The fusion index data were expressed relative to the forskolin-untreated WT control.
9
Forskolin-Induced BeWo Cell Syncytialisation
10
Quantifying T-Tubule Structure in Cardiomyocytes
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Quantification of t-tubule structure in isolated ventricular and atrial myocytes was performed by staining cells with 10 µmol/L di-8-ANEPPS (Invitrogen, Paisley, United Kingdom) or with CellMask Orange (1:1000 dilution; Thermo Fisher Scientific, Waltham, MA; C10045). For each cell, t-tubule density was determined by thresholding the image intensity of the entire cell by the Otsu method, using an automated algorithm in ImageJ (National Institutes of Health). The t-index17 (link) was then calculated for the myocyte interior, defined as the percentage of the cellular cross-sectional area, excluding the nucleus, occupied by above-threshold pixels. Cells exhibiting a t-index of ≥ 2% were defined as being tubulated.12 (link) Detubulation was performed using a protocol similar to that described by Kawai et al.18 (link)
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