DiBAC4(3) (Sigma), a dye with Em-sensitive uptake (Epps et al., 1994 (link)), was used to measure Em for cells incubated in various media. Confluent NIH-3T3 cells were serum-starved in DMEM for 4–6 hours, after which the cells were detached by trypsinization, were washed, and the cell suspension was incubated with DiBAC4(3) (400nM), for 30 minutes at 37°C. The cells were then analyzed on a FACSCalibur flow cytometer (BD Biosciences), using 488nm laser excitation and a 530/30 emission filter. Media with various K+ concentrations were used as positive controls for membrane depolarization. A standard curve for DiBAC4(3) fluorescence was determined by permeabilizing cells with 2μg/mL gramicidin A (Sigma), incubating them with DiBAC4(3) (0, 25, 50, 100, 200, 400nM), followed by fluorescence measurement by flow cytometry.
Dibac4 3
Manufactured by Merck Group
Sourced in United States, Germany
DiBAC4(3) is a fluorescent dye commonly used in laboratory research. It is a membrane-permeable, bis-oxonol dye that exhibits increased fluorescence upon binding to the intracellular environment. DiBAC4(3) is often utilized to assess changes in membrane potential and monitor cellular activity.
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Lab products found in correlation
Dibac4 3, by Dojindo Laboratories (2 mentions)
Ns1619, by Merck Group (2 mentions)
H2dcfda, by Thermo Fisher Scientific (2 mentions)
Nucleocounter nc 3000, by ChemoMetec (1 mentions)
Fluostar optima microplate reader, by BMG Labtech (1 mentions)
Gramicidin a, by Merck Group (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Sytox green, by Thermo Fisher Scientific (1 mentions)
Ckx53 fluorescence microscope, by Olympus (1 mentions)
Facscalibur, by BD (1 mentions)
Accuri c6, by BD (1 mentions)
Hepes, by Merck Group (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
5 cfda, by Merck Group (1 mentions)
Dcfh da, by Merck Group (1 mentions)
Fluorescence spectrophotometer, by Agilent Technologies (1 mentions)
Black opaque 96 well plate, by Corning (1 mentions)
Infinite m200 microplate reader, by Tecan (1 mentions)
Spectramax id3, by Molecular Devices (1 mentions)
27 protocols using dibac4 3
1
Membrane Potential Measurement by DiBAC4(3)
2
Measuring Cell Membrane Potential with DiBAC4(3)
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Cell membrane potential was measured using the fluorescence dye DiBAC4(3) (Dojindo, Japan). HUVECs were seeded on a confocal dish at a density of 4 × 104 cells/well. After culturing for 24 h, the cells were washed with DPBS, and then incubated with 5 μmol/L DiBAC4(3) diluted with 20 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Sigma Aldrich, USA) for 30 min at 37 °C, in the dark. Subsequently, BTO nanoparticles were added and LIPUS treatment (10 s) was performed. Fluorescence images were obtained using CLSM.
3
Membrane Depolarization Monitoring in Salicornia and Arabidopsis
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PM depolarization was monitored using the voltage-sensitive fluorescent probe DiBAC4(3) (Sigma-Aldrich; Konrad and Hedrich 2008 (link)), which permeates the cell membrane and accumulates in the cytosol upon membrane depolarization. DiBAC4(3) has a fluorescence intensity in the aqueous solution that increases several folds in a milieu like a cytoplasm (Br�uner et al. 1984) (link). Salicornia europaea suspension cells of SePSS-RNAi and the EV controls were incubated in the dark for 15 min with 1 μM DiBAC4(3) in a medium containing 0, 400 and 800 mM NaCl. Cells were maintained under shaking during the incubation. Fluorescence was detected and imaged under a confocal microscope (LSM-510). The fluorophore was excited at 488 nm, and the emission was detected between 500 and 550 nm. The average fluorescence intensity of cells was calculated by ImageJ 1.49 (Image Processing and Analysis in Java, http://imagej.net ). Cells from at least 10 microscope fields under 20� objective were analyzed.
For assays in Arabidopsis, sterilized seeds of WT and transgenic lines were sown on a medium containing 1/2 MS salts, with 1% (w/v) sucrose and 1% (w/v) agar, stratified at 4�C in the dark for 2 d. Roots of 4-day-old seedlings were incubated in the dark for 15 min with 1 μM DiBAC4(3) in a medium containing 0 and 200 mM NaCl. At least 12 plants were analyzed.
For assays in Arabidopsis, sterilized seeds of WT and transgenic lines were sown on a medium containing 1/2 MS salts, with 1% (w/v) sucrose and 1% (w/v) agar, stratified at 4�C in the dark for 2 d. Roots of 4-day-old seedlings were incubated in the dark for 15 min with 1 μM DiBAC4(3) in a medium containing 0 and 200 mM NaCl. At least 12 plants were analyzed.
4
Yeast Cell Membrane Integrity Assay
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S. cerevisiae cells were grown to mid-log phase, pelleted at 2,000 x g for 2 minutes and washed twice in buffer A (100 mM MOPS and 1 mM KCl adjusted with Trizma base to pH 7.0). Cells were then resuspended in buffer A to an OD600 of 0.2. For each well 50 μL of buffer A with 3.3 μg/mL propidium iodide (PI) (Thermo Fisher Scientific, catalog number P1304MP) and 1 μg/mL DiBAC4(3) (Sigma-Aldrich, catalog number D8189)) was mixed with 50 μL S. cerevisiae cell suspension and transferred to a 96 well plate containing 1 μL of inhibitor (in DMSO). A lysis control was performed with Reagent Y100 (ChemoMetec). Five thousand cells were measured per experiment with an exposure time of 1000 ms using a NucleoCounter NC-3000 (ChemoMetec). Dark field was used as the masking channel to select the yeast cells, and the DiBAC4(3) (ex: 530 nm, em 675/75 nm) and PI (ex 630 nm, em 740/60 nm) channels were used to measure the membrane potential and membrane integrity, respectively. Only cells with a pixel size of 10–40 were included in the analysis, to avoid analysis of non-cell artifacts.
5
Measuring MRSA Membrane Potential
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DiBAC4(3) (Sigma-Aldrich, St. Louis, MO, USA) was used to detect the membrane potential of MRSA. MRSA cells were cultured and washed as described above. DiBAC4(3) (0.1 μg/μL; 1 μL) was added to 1 mL diluted cells for staining. The cells were then incubated at 25°C for 30 min in the dark. After staining, the samples were pelleted, the supernatant removed, and the cells suspended in 500 μL PBS. Finally, the samples were analyzed for the fluorescence intensity of FITC-DiBAC4(3) using flow cytometry.
6
Membrane Depolarization by hLF1-11 Peptide
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Bacterial cells (107 CFU/mL), harvested in mid-log-phase, were incubated in 1:16 diluted MHB with various concentrations of hLF1-11 for 1 h at 37°C. At the same time, samples were stained with 10 μg/mL DiBAC4(3) (Sigma-Aldrich) added from a stock solution of 5 mg/mL in DMSO. The hLF1-11 peptide was added at 44 μg/mL (2× MIC) to assess membrane depolarization at different time points for up to 1 h. Untreated cells were used as the negative control, and CCCP as the positive control. The fluorescence emission of DiBAC4(3) was detected as green fluorescence using 485 excitation/520 emission optical filters on a FLUOstar OPTIMA Microplate Reader (BMG LABTECH, Ortenberg, Germany). The depolarizing activity of the hLF1-11 peptide was evaluated as decreased fluorescence in the medium after uptake of DiBAC4(3).
7
Monitoring Chloride Homeostasis Dynamics
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Cells were treated with 20 μm DiBAC4(3) (Sigma) in medium for 20 min at 37 °C and protected from light. Conditions were selected as optimal from standardization of DiBAC4(3) concentrations, incubation time, and temperature. Following labeling, cells were washed and either mock-infected or infected with BUNV (MOI 1). Cells were treated with TEA (10 mm ) or quinidine (200 μm ) as controls. The average mean intensity of green fluorescence GCU (Green Calibration Units per well) was measured from several widefield images using IncuCyte ZOOM live cell imaging at 1-h intervals for up to 6 h. Values are presented as the mean fluorescent intensity per cell normalized to untreated controls from a minimum of three independent experiments. Assays of [Cl−]i using 5 mm 6-methoxy-quinolyl acetoethyl ester (MQAE) were performed as previously described (22 (link)). 5-Nitro-1–3-phenylpropylamino benzoic acid (NPPB) and indyanyloxyacetic acid 94 (IAA-94) were included as compounds known to modulate Cl− function for assay verification.
8
Membrane Potential and Caspase Assays for K. pneumoniae
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DiBAC4(3) (Sigma-Aldrich, St. Louis, MO, USA) was used to detect the membrane potential of K. pneumoniae. K. pneumoniae were cultured and washed as described above. DiBAC4(3) (0.1 µg/µL, 1 µL) was added to 1 mL diluted cells for staining. The cells were then incubated in darkness at room temperature for 30 min. After staining, the FITC-DiBAC4(3) fluorescence intensity of the samples was analyzed by flow cytometry.
Analysis of bacterial proteins with caspase-like substrate affinity. FITC-conjugated pan-caspase inhibitor peptide, Z-V-d-FMK (Intracellular Caspase Detection ApoStat, USA), was used to detect whether platelet-K. pneumoniae coculture supernatant could induce the expression of bacterial proteins that can bind to caspase substrate peptides. The cells of K. pneumoniae were cultured and washed as described above. Then, 10 µL Z-V-d-FMK-FITC was added into 1 mL diluted cells and incubated at 37°C in the dark for 30 min. Next, the cells were washed with PBS and suspended with 500 µL PBS. Finally, the fluorescence intensity of Z-V-d-FMK-FITC was analyzed by flow cytometry.
Analysis of bacterial proteins with caspase-like substrate affinity. FITC-conjugated pan-caspase inhibitor peptide, Z-V-d-FMK (Intracellular Caspase Detection ApoStat, USA), was used to detect whether platelet-K. pneumoniae coculture supernatant could induce the expression of bacterial proteins that can bind to caspase substrate peptides. The cells of K. pneumoniae were cultured and washed as described above. Then, 10 µL Z-V-d-FMK-FITC was added into 1 mL diluted cells and incubated at 37°C in the dark for 30 min. Next, the cells were washed with PBS and suspended with 500 µL PBS. Finally, the fluorescence intensity of Z-V-d-FMK-FITC was analyzed by flow cytometry.
9
Determination of S. aureus Membrane Potential
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The determination of S. aureus (ATCC29213) cell membrane potential was based on the method of Guo et al. [37 (link)], with some minor modifications. An amount of 200 μL of the bacterial suspension was added to a 96-well black microtiter plate, followed by addition of juglone to final concentrations of 2 × MIC, MIC, and 0 (the corresponding volume of DMSO was added as a control), respectively. The microtiter plate was placed at 37 °C, and after 1 h of incubation, DIBAC4(3) (Sigma-Aldrich, St. Louis, MO, USA) was added to each well so that its final concentration was 1 μM. After a 10-min incubation in the dark, the fluorescence intensity of all samples were measured on a microplate reader (spark, Austria) (excitation wavelength was 492 nm, emission wavelength was 515 nm). The fluorescence values measured by different concentrations of juglone solution (without bacterial suspension) were used as the background fluorescence values. The actual fluorescence values of different experimental treatment groups was the difference between the measured value and the corresponding background fluorescence value.
10
Assessing Cell Membrane Potential
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The cell membrane potential was assessed by staining the cells with Bis-(1,3-Dibutylbarbituric Acid) Trimethine Oxonol (DiBAC4(3), Sigma–Aldrich), as described by Teixeira-Santos et al. (2012) (link). The fluorescence intensity (FI) at FL1 (fluorescent detector; 530 nm) was registered and a staining index (SI) was defined as the ratio between the FI of treated cells and the FI of non-treated cells.
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