Through immunofluorescence staining, we evaluated mitochondrial membrane potential (MMP) and ROS levels in the macrophages isolated from the peritoneal cavity of the experimental and WT mice. As in our previous study [18 (link)], the ROS level was estimated using dichlorofluorescein diacetate (Molecular Probes, Eugene, OR, USA) as an oxidative fluorescent probe, whereas MMP was estimated using the lipophilic cationic fluorescent dye 3,3′-dihexyloxacarbocyanine iodide (Sigma-Aldrich). The mean fluorescent intensity was analyzed using a flow cytometer (FACS Calibur; BD Biosciences PharMingen, San Diego, CA, USA).
3 3 dihexyloxacarbocyanine iodide
Manufactured by Merck Group
Sourced in United States
3,3′-dihexyloxacarbocyanine iodide is a fluorescent dye used in laboratory applications. It functions as a membrane potential indicator, enabling the monitoring of changes in membrane potential in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Merck Group (2 mentions)
Facscalibur, by BD (1 mentions)
Dichlorofluorescein diacetate, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 405, by Thermo Fisher Scientific (1 mentions)
Matlab, by MathWorks (1 mentions)
Cell imaging cover slips, by Eppendorf (1 mentions)
Lsm 780 confocal microscope, by Zeiss (1 mentions)
Alexa 594 anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Slowfade gold, by Thermo Fisher Scientific (1 mentions)
Anti lamin b1 antibody, by Abcam (1 mentions)
6 protocols using 3 3 dihexyloxacarbocyanine iodide
1
Mitochondrial and ROS Profiling in Macrophages
2
Evaluating Bacterial Biofilm Susceptibility
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To assess the ability of the extract to kill bacterial cells in biofilms, confocal laser scanning microscopy was performed. For that, bacteria were grown in BM medium in a cell imaging chambered coverslip with 8 wells (Ibidi, Gräfelfing, Germany) under static conditions. After 24 h of incubation, fresh broth supplemented with extract with a final concentration equal to 2 × MIC was added. Chlorhexidine was used as the reference antiseptic. After 3 h of incubation, biofilms were stained for 15 min with 3,3′-Dihexyloxacarbocyanine iodide (Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 0.02 µg/mL (green fluorescence) and propidium iodide (Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 3 µg/mL (red fluorescence) to differentiate live and dead cells. CLSM was performed using an Olympus IX83 (Olympus Europa, Hamburg, Germany) inverted microscope supplemented with a STEDYCON ultrawide extension platform.
3
Platelet Adhesion and Fibrin Formation Kinetics
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Fresh blood was reconstituted with PLTs from the PC aiming for an average of 40% hematocrit and 250 × 109 PLTs/L. Sample preparation and fluorescent labeling with 3,3′‐dihexyloxacarbocyanine iodide (Sigma‐Aldrich) and blue fluorescent dye (Alexa Fluor 405, Life Technologies) was as published before.22 , 23 , 27 Adhesion of PLTs onto collagen and formation of fibrin was studied by monitoring changes in median fluorescence of respective fluorophores as a function of time during perfusion of the reconstituted and recalcified blood at a wall shear rate of 1000/sec as described previously.27 PLT adhesion rate (/sec) indicates the rate of PLT deposition. The variables retrieved for fibrin deposition included coagulation rate (/sec), which is the linear portion of fibrin deposition kinetics and coagulation onset (sec). This is the lag time indicating the moment of coagulation onset defined as the intercept with the x‐axis of the extrapolated linear regression of the thrombus formation by fibrin fluorescence. This analysis takes into account thrombus growth in the z‐plane. The outcome variables were extracted from the raw fluorescence data using a software plugin developed in computer software (MatLab, MathWorks).
4
Mitochondrial Membrane Potential Assay
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The MMP (ΔΨm) of HepG2 cells treated with green synthesized monometallic and bimetallic NPs was taken using 3,3′-dihexyloxacarbocyanine iodide (Sigma), which works by staining mitochondria according to their MMP. Cells were incubated in culture media supplied with 25 nM 3,3′-dihexyloxacarbocyanine iodide at 37 °C for 40 min. The MMP was expressed as relative fluorescent units (RFU). The experiment was performed thrice.
5
Biofilm Viability Assay by CLSM
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To evaluate the viability of biofilm-embedded cells, bacterial suspension was inoculated in BM broth and grown on cell imaging cover slips (Eppendorf) under static conditions. After 48 h of growth, half of the medium was exchanged by the fresh medium. Next Ficin and antimicrobials were added as described previously and further incubated for 24 h. The samples were then stained for 5 min with the 3,3′-Dihexyloxacarbocyanine iodide (Sigma) at final concentration of 0.02 μg/ml (green fluorescence) and propidium iodide (Sigma) at final concentration of 3 μg/ml (red fluorescence) to differentiate between bacteria with intact and damaged cell membranes (live and dead cells). Confocal laser scanning microscopy images (CLSM) were obtained with a Carl Zeiss LSM 780 confocal microscope with Ζ-series images taken in 1-μm slices.
6
Nuclear Envelope and Replication Visualization
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For detecting the NE, each 2-μl extract sample was gently mixed on a glass slide with 4 μl of the fixing solution (3.7% formaldehyde, 2 μg/ml Hoechst 33258, 50% glycerol, 80-mM KCl, 15-mM NaCl, 15-mM Pipes-KOH, pH 7.2) containing 10 μM of 3,3′-dihexyloxacarbocyanine iodide (Sigma-Aldrich) and squashed under a 22-mm × 22-mm square coverslip.
For detecting replication activity and immunofluorescence, each 10 μl extract sample was diluted with 90-μl of extraction buffer (EB) (100-mM KCl, 2.5-mM MgCl2, 50-mM Hepes-KOH, pH 7.5), 11 μl of 37% formaldehyde was then added, incubated at RT for 10 min, 1 ml of EB was added further, which was loaded into a swinging bucket for collecting suspension culture cells (CS-2, Tomy). Nuclei were collected by centrifuge (500g, 5 min) onto poly-L-lysine–coated coverslip (IWAKI) through 0.5-ml of EB plus 30% sucrose layer. DNA replication was labeled with 1 μM of CF594-dUTP (Biotium). Nuclear lamin B1 was detected by sequential incubation with anti-lamin B1 antibody (ab16048, Abcam) and Alexa 594 anti-rabbit IgG (Thermo Fisher). The coverslips were washed with TBS-T, PBS, and dH2O and mounted on glass slides with 3 μl of SlowFade Gold (Thermo Fisher) containing 2 μg/ml Hoechst 33258.
For detecting replication activity and immunofluorescence, each 10 μl extract sample was diluted with 90-μl of extraction buffer (EB) (100-mM KCl, 2.5-mM MgCl2, 50-mM Hepes-KOH, pH 7.5), 11 μl of 37% formaldehyde was then added, incubated at RT for 10 min, 1 ml of EB was added further, which was loaded into a swinging bucket for collecting suspension culture cells (CS-2, Tomy). Nuclei were collected by centrifuge (500g, 5 min) onto poly-L-lysine–coated coverslip (IWAKI) through 0.5-ml of EB plus 30% sucrose layer. DNA replication was labeled with 1 μM of CF594-dUTP (Biotium). Nuclear lamin B1 was detected by sequential incubation with anti-lamin B1 antibody (ab16048, Abcam) and Alexa 594 anti-rabbit IgG (Thermo Fisher). The coverslips were washed with TBS-T, PBS, and dH2O and mounted on glass slides with 3 μl of SlowFade Gold (Thermo Fisher) containing 2 μg/ml Hoechst 33258.
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