DiBAC4(3) (Molecular Probes, Sigma), a membrane potential-sensitive fluorescent probe, was used for membrane potential determination [22] (link). MRSA P-1 cells were cultured to the logarithmic phase and harvested by centrifugation, and then the precipitates were washed and adjusted to OD600 nm = 0.5. DiBAC4(3) solution was added to the cell suspensions and the final concentration was 0.1 µM. After 30 min incubation, the mixtures were added with GEON (final concentrations of 0, 1/2MIC, 1MIC, 2MIC, 4MIC) and placed at dark for 3 h. the fluorescence of each sample was determined using a fluorescence microplate reader (Thermo Scientific, Germany) at excitation/emission wavelengths of 492/515 nm.
Dibac4 3
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada, Germany
DiBAC4(3) is a membrane-permeable dye that binds to intracellular proteins and can be used to assess changes in membrane potential in cells. It exhibits increased fluorescence upon binding to proteins.
Automatically generated - may contain errors
Lab products found in correlation
Fura 2 am, by Thermo Fisher Scientific (3 mentions)
Infinite m200 pro, by Tecan (3 mentions)
Synergy h1, by Agilent Technologies (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Cary eclipse fluorescent spectrophotometer, by Agilent Technologies (2 mentions)
Fv 1000 mpe spectra, by Olympus (2 mentions)
Dibac4 3, by BD (2 mentions)
Spectramax m2, by Molecular Devices (2 mentions)
Facscalibur, by BD (2 mentions)
Fluorescence microplate reader, by Thermo Fisher Scientific (1 mentions)
Ouabain, by Merck Group (1 mentions)
Fm4 64 dye, by Thermo Fisher Scientific (1 mentions)
Amiloride, by Merck Group (1 mentions)
Carbonyl cyanide 3 chlorophenylhydrazone cccp, by Merck Group (1 mentions)
Glibenclamide, by Merck Group (1 mentions)
Tram 34, by Merck Group (1 mentions)
Rotenone, by Merck Group (1 mentions)
Apamin, by Bio-Techne (1 mentions)
Antimycin, by Merck Group (1 mentions)
Xe991, by Bio-Techne (1 mentions)
78 protocols using dibac4 3
1
Membrane Potential Determination of MRSA
2
Larval Tissue Dissection and Imaging
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Larvae were washed with 70% EtOH and PBS prior to dissection. Live tissue was dissected in Schneider's media (#21720001, Gibco), and care was taken to not damage or stretch tissue. For DiBAC staining, imaginal discs were incubated in 1.9µM DiBAC4(3) (bis-(1,3-dibutylbarbituric acid) trimethine oxonol; DiBAC4(3); Molecular Probes)) in Schneider's media for 10 minutes with gentle rotation. A small amount media was used to mount the discs, such that addition of a coverslip did not destroy the tissue, and discs were imaged right away. 100µM amiloride (#A7419, Sigma-Aldrich) and 100µM ouabain (#O3125, Sigma-Aldrich) were added to DiBAC solution for pharmacology experiments. Discs imaged in FM4-64 dye (#T13320, ThermoFisher) were incubated in 9µM FM4-64, and imaged without washing, to preserve staining of the cell membrane. Discs expressing ArcLight were dissected, mounted, and imaged in Schneider's.
For optogenetics experiments, carcasses were dissected and cleaned (fat body removed) in Schneider's medium. Carcasses were loaded onto a glass cover slip in a large drop of Schneider's, and either kept in the dark (control condition) or exposed to activating light (480nm for ChR2 and ChloC experiments, 647nm for ReaChR experiments). After exposure, carcasses were immediately fixed and prepared for immunohistochemistry.
For optogenetics experiments, carcasses were dissected and cleaned (fat body removed) in Schneider's medium. Carcasses were loaded onto a glass cover slip in a large drop of Schneider's, and either kept in the dark (control condition) or exposed to activating light (480nm for ChR2 and ChloC experiments, 647nm for ReaChR experiments). After exposure, carcasses were immediately fixed and prepared for immunohistochemistry.
3
Dual Live Cell Imaging Techniques
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We used two different dual live cell imaging techniques in this study. Dual calcium and sodium imaging was performed as previously described (Dutta Banik et al., 2018 (link)). Briefly, isolated cells were simultaneously loaded with Fura 2-AM (ThermoFisher) and Asante NaTrium-2 (TEFLabs, Inc., Austin, TX). For the dual calcium and membrane potential imaging, cells were simultaneously loaded with Fura 2-AM and DiBAC4(3) (ThermoFisher Scientific). In both sets of experiments, cells were excited at 340 nm, 380 nm, and 488 nm excitation wavelengths. The dual live cell images were captured every 4 s using a multiedge dichroic beam-splitter that captures emissions at both 510 nm and 540 nm using Imaging Workbench (Indec Biosystems). Experiments were graphed and analyzed using Origin software.
4
Mitochondrial Respiration and Ion Channel Inhibitors
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DiBAC4(3) was obtained from Thermo Fisher Scientific. Rotenone, antimycin, oligomycin B and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (all from Sigma-Aldrich, Dorset, UK) were dissolved in DMSO as 1, 1, 6 and 10 mM stock solutions, respectively. K+ channels inhibitors, glibenclamide (Sigma-Aldrich), penitrem A (Alomone labs, Jerusalem, Israel), tram34 (Sigma-Aldrich), apamin (Tocris Bioscience, Abingdon, UK) and XE991 (Tocris Bioscience) were also prepared in DMSO as 10, 1, 10, 1 and 10 mM stock solutions, respectively. Other chemicals were obtained from Sigma-Aldrich or VWR (Leicestershire, UK).
5
Evaluating Bacterial Membrane Integrity and Fluidity
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As vesicle production can be associated with extensive cell lysis, cell death was quantified by measuring the fluorescent signal in the cell population using propidium iodide (Sigma-Aldrich, P4864), a membrane-impermeable nucleic acid stain commonly used as a cell death marker. Specifically, red fluorescence is indicative of cellular loss of membrane impermeability. On the other hand, membrane depolarization was quantified by the emission of a green fluorescence signal using voltage-dependent dye DIBAC4(3) (Thermo Fisher Scientific B438). Microscopy snapshots were taken to visualize the population of bacteria under these various dye treatments. Membrane fluidity was assessed by using the Membrane Fluidity Kit (Abcam ab189819), which measures the changes in fluorescence spectral properties of lipid analog probes added to the cell culture. Fluorescence shifts (400 to 470 nm) resulting in changes in membrane viscosity were read in a TECAN microplate reader (Infinite M200 PRO). The ratio of emission at 470 nm to emission at 400 nm was normalized to that of unlabeled cell conditions. In all assays, total fluorescence was normalized to biomass (optical density at 600 nm) in each sample.
6
Wound Closure Assay with Chemical Inhibitors
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Cells were seeded onto Petri dishes at a density of 5 × 105 cells per dish and grown to confluency. Scratch wounds were made with a 200 μL pipette tip, after which detached cells were removed by washing with phosphate-buffered saline. In order to inhibit cell proliferation, culture media was replaced to serum-free DMEM. Compounds 4d , 4e , 4f , and 4h were added to the cultures at a dose of 10 μg/mL and incubated for 24 h. After that, the cells were stained with Hoechst 33,342 (Thermo Fisher Scientific, Waltham, MA, USA), by adding 2 μL of 1 mg/mL stock solution to 2 mL of medium and DIBAC4 (3) (Thermo Fisher Scientific, Waltham, MA, USA) at the same dose. Images were captured using confocal microscope (Axio Observer Z1, Carl Zeiss MicroImaging GmbH, Jena, Germany). The percentage of wound closure in five randomly chosen fields was calculated with NIH ImageJ software.
7
Measuring Plasma Membrane Potential
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To investigate the plasma membrane potential, we used the membrane potential‐sensitive dye DiBAC4(3) (Thermo Fisher Scientific, #B438). Wing discs from up‐crawling third‐instar larvae were dissected in Grace's medium supplemented with 5% FBS and 20 nM 20‐hydroxyecdysone (full medium) (Dye et al, 2017 ), and incubated with 500 nM DiBAC4(3) in full medium for 15 min at 29°C. After washing twice with full medium, wing discs were mounted and imaged immediately. A higher fluorescence intensity of DiBAC4(3) indicates depolarization of the plasma membrane.
8
Measuring Acute Membrane Potential Changes
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Acute changes in the plasma membrane potential were measured by fluorescent microscopy using fluorescent dye that are sensitive to membrane potential FluoVoltTM and DiBAC4(3) (Thermofischer, Saint Aubin, France) as described by the manufacturer. To assess the effects of total membrane disruption oubain 10 nM or 10 μM valinomycin was added to the cell culture medium. MATLAB software was used to calculate pixel intensities.
9
Evaluating Compound-Induced Cell Changes
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Cells were seeded in Petri dish, 5 × 105 cells per dish and cultivated until 100% confluence. Then the cell monolayer was scraped in straight line with a 200 μL pipet tip. Full culture media was replaced to serum-free DMEM for inhibition of cell proliferation and cells were treated with 4a (10μg/mL) and 5a (20 μg/mL) compounds and incubated 24 h. After that cells were stained with Hoechst 33342 (ThermoScientific, Waltham, MA, USA, 2 μL of 1 mg/mL stock solution to 2 mL of medium) and DIBAC4 (3) (ThermoScientific, Waltham, MA, USA, 2 μL of 1 mg/mL stock solution to 2 mL of medium) for contrasting and evaluated at confocal microcope.
10
Membrane Depolarization Assay for Antimicrobial Peptides
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To get hints for membrane damages we used approximately 1.5 x 106 CFU log-phase bacteria in a final volume of 95 μl TSB (1:6 diluted in H2O). We added peptides in concentrations 50, 75 and 95 μg/ ml in a final volume of 10 μl and incubated these suspensions for 1h at 37°C. Subsequently, 1 μg/ml of the membrane potential sensitive dye DiBAC4(3) [bis-[1,3-dibutylbarbituric acid) trimethine oxonol] (Thermo Fisher Scientific, USA) was added and incubated for 10 min at room temperature. Then, bacteria were centrifuged (5 min at 4°C and 7000 rpm) and re-suspended in 300 μl PBS. The percentage of depolarized fluorescent bacteria was determined using a FACSCalibur flow cytometer (BD, Sparks, USA), as described earlier [23 (link)]. Experiments were repeated at least three times and mean +/- SEM is shown.
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