Dioc6

Manufactured by Thermo Fisher Scientific

Sourced in United States, Spain, Italy

DiOC6 is a fluorescent dye used for the detection and analysis of mitochondrial membrane potential in cells. It is a lipophilic cationic dye that accumulates in active mitochondria due to the negative membrane potential. DiOC6 can be used in flow cytometry, fluorescence microscopy, and other fluorescence-based techniques to assess mitochondrial function and health.

Automatically generated - may contain errors

Lab products found in correlation

109 protocols using dioc6

Identifying Non-Apoptotic AnV+ B Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

To show that AnV+ B cells are not only apoptotic cells, several methods were employed. We first used DAPI (Sigma-Aldrich, Saint-Quentin, France) on B cells sorted by FACSARIA according to their AnV staining after 72 hr of culture, to assess the numbers of dead cells. We also used DiOC6 (LifeTechnologies) on B cells sorted by FACSARIA according to their AnV staining, to assess the cells in early apoptosis. Briefly, cells were incubated with DiOC6(3) diluted at 40nM in culture media (1M°cells/ml of dye) protected from light during 30 min at 37°C.
We also used propidium iodide (PI) (LifeTechnologies) to analyze by flow cytometry cell cycle in B cells according to PS exposure level. This allow quantification of the cells in the subG1 phase, corresponding to an apoptotic state. Briefly, PBMC, stained as previously described using anti-19 antibodies and annexin V for extracellular staining, were fixed in Cytofix/Cytoperm buffer for 30 min on ice (BD Biosciences, Le pont-de-Claix, France) and incubated 30min at room temperature in 1X Perm/wash Buffer, BD Biosciences) with addition of PI (50μg/ml) and RNAse A (100μg/ml).

Audo R., Hua C., Hahne M., Combe B., Morel J, & Daien C.I. (2017). Phosphatidylserine Outer Layer Translocation Is Implicated in IL-10 Secretion by Human Regulatory B Cells. PLoS ONE, 12(1), e0169755.

+ Open protocol

+ Expand

Quantification of Apoptosis by Multiple Techniques

Check if the same lab product or an alternative is used in the 5 most similar protocols

Apoptosis was measured from in vitro cultures described above by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), annexin-propidium iodide (PI), and 3,3′-dihexyloxacarbocyanine iodide (DiOC6) methods [24 ]. TUNEL was measured using a Fluorescein In Situ Cell Death Detection Kit from Roche (Indianapolis, IN) as per manufacturer’s instructions. Annexin-PI staining was performed by using a FITC Annexin V kit purchased from Biolegend (San Diego, CA) as per manufacturer’s instructions. DiOC6 was purchased from Molecular Probes (Eugene, OR), and cells were stained with 40nM of DiOC6 for 30 minutes at 37°C before analysis. For studies using caspase inhibitors, inhibitors for caspases-2, 3, 8, and 9 were purchased from Calbiochem (San Diego, CA) and incubated simultaneously with the various treatments described above at a concentration of 100µM. Cells from all these assays were analyzed using flow cytometry to detect apoptosis.

Busbee P.B., Nagarkatti M, & Nagarkatti P.S. (2015). Natural Indoles, Indole-3-Carbinol (I3C) and 3,3’-Diindolylmethane (DIM), Attenuate Staphylococcal Enterotoxin B-Mediated Liver Injury by Downregulating miR-31 Expression and Promoting Caspase-2-Mediated Apoptosis. PLoS ONE, 10(2), e0118506.

+ Open protocol

+ Expand

Cytotoxicity Assay of FD-895 and Pladienolide B

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Normal PBMCs were treated with FD-895 (100 nM to 2.0 μM), and pladienolide B (100 nM to 2.0 μM), for 48 h. Cell viability was determined by flow cytometry after staining with conventional live staining with 40 μM 3,3′dihexyloxacarbocyanine iodide (DiOC₆; Life Technologies, Carlsbad, CA, USA) and 15 μM (10 μg/mL) of propidium iodide (PI; Sigma-Aldrich, St Louis, MO, USA). Data were analyzed by using FlowJo software (version 6.4.7; Tree Star). Using this assay, viable cells excluded PI and stained brightly positive for DiOC₆ as it targets metabolically active mitochondria of alive cells [30 (link)–33 (link)].

Kumar D., Kashyap M.K., Yu Z., Spaanderman I., Villa R., Kipps T.J., La Clair J.J., Burkart M.D, & Castro J.E. (2022). Modulation of RNA splicing associated with Wnt signaling pathway using FD-895 and pladienolide B. Aging (Albany NY), 14(5), 2081-2100.

+ Open protocol

+ Expand

Microfluidic Platelet Labeling and Imaging

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

PDMS microfluidic devices were produced using standard soft lithography techniques and bonded to glass substrates. CTC-iChips are produced by a third party^{28 (link)} using medical grade cyclic olefin copolymer (COC). The devices were primed with 70% ethanol followed by PBS containing 0.2% (w/v) Pluronic F-68 before blood processing. For time-lapse experiments, whole blood was incubated with DiOC6 (1 μM; Life Technologies) for 10 minutes to label platelets, before it was loaded into a syringes (BD) and processed in the device using a syringe pump (Harvard Apparatus PHD 2000) at the indicated flow rates. Where indicated, GSH (30 mM; Sigma), NAC (30 mM; Sigma), or EDTA (4 mM; Ambion) were added to blood immediately prior to blood processing. Fluorescence images were acquired at 30-second intervals with a QImaging Retiga 2000R camera using a Nikon Plan Fluor 4×/0.13 objective on a Nikon Eclipse 90i microscope. The area-averaged fluorescence intensity of each image was measured (using NIS Elements ver. 3.22) in a rectangular region-of-interest (ROI) that includes all microfluidic pillars in the device within the image. The ROIs for PDMS devices and CTC-iChips measured 2940 μm by 1740 μm and 2900 μm by 2160 μm, respectively. Fluorescence images were pseudocolored using Fiji for easy visualization.

Wong K.H., Edd J.F., Tessier S.N., Moyo W.D., Mutlu B.R., Bookstaver L.D., Miller K.L., Herrara S., Stott S.L, & Toner M. (2018). Anti-thrombotic strategies for microfluidic blood processing. Lab on a chip, 18(15), 2146-2155.

+ Open protocol

+ Expand

Screening for Nuclear-Cytoplasmic Ratio Mutants

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

The 3-stage screen was carried out as described above. Firstly, deletion mutants were incubated at 25°C for 12 to 20h in 300 μl of YE4S in 96-well plates then inoculated onto YE4S agar plates containing DiOC₆ at 10 μg/ml (Life technologies) using a pin tool (V & P Scientific, Inc) and incubated at 25°C for 12 to 20h. The N/C ratio of each mutant strain was estimated by comparison with the wild type strain growing in the same plate using a Zeiss Axioskop 40 microscope. 102 of the 2,969 deletion mutants failed to grow on plates so were excluded from the screen. 366 mutant strains were selected for a secondary visual screen in liquid medium. Cells were collected from individual exponentially growing cultures of the 366 candidate mutants, stained with DiOC₆ and visually screened to estimate the N/C ratio. The 97 strains selected for the tertiary screen were tagged with the nuclear envelope marker protein Cut11-GFP and the nuclear and cellular volumes measured to assess the N/C ratio [5 (link)]. Images were analysed using ImageJ (NIH) as previously described [5 (link)]. Nuclear volume of shape mutants was calculated using ImageJ.

Kume K., Cantwell H., Neumann F.R., Jones A.W., Snijders A.P, & Nurse P. (2017). A systematic genomic screen implicates nucleocytoplasmic transport and membrane growth in nuclear size control. PLoS Genetics, 13(5), e1006767.

+ Open protocol

+ Expand

Characterization of Apoptosis in CLL B-cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cell viability was determined after the indicated treatments in a standard MTT assay, as per the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO). Apoptosis was determined by Annexin V and propidium iodide staining, per the manufacturer’s instructions (BioLegend, San Diego, CA), after gating on CD5⁺CD19⁺ CLL B-cells. Loss of the mitochondrial membrane potential in apoptotic cells was determined by 3,3′-dihexyloxacarbocyanine iodide (DiOC₆, Life Technologies, Grand Island, NY) staining. Briefly, cells were loaded with 40 nM DiOC₆ 15 minutes prior to flow cytometric analysis. Viable, non-apoptotic cells were identified as DiOC₆^high. Cell and nuclear morphology following the indicated treatments was visualized by phalloidin and DAPI labeling. Briefly, macrophages or NLC cells were harvested by vigorous pipetting and fixed in 4% paraformaldehyde. Fixed cells were spun at 2000 rpm for 3 minutes using a Cytopro cytocentrifuge (Wescor, Logan, UT) unto Colorfirst Plus microscope slides (Fisherbrand, Pittsburgh, PA). Cells were labeled with phalloidin (1:200;) and DAPI, and preserved with ProLong Gold (Life Technologies) anti-fading reagent prior to visualization by immunoflourescent microscopy, using an Olympus microscope (200x).

Polk A., Lu Y., Wang T., Seymour E., Bailey N.G., Singer J.W., Boonstra P.S., Lim M.S., Malek S, & Wilcox R.A. (2016). Colony-stimulating Factor-1 Receptor is Required for Nurse-like Cell Survival in Chronic Lymphocytic Leukemia. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(24), 6118-6128.

+ Open protocol

+ Expand

Membrane Staining and Fluorescence Quantification

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells (10⁵) from overnight cultures of wild-type (WT), mutant, and complement strains were washed and resuspended in phosphate-buffered saline (PBS; Gibco), mixed with 200 nM DiOC₆ (Life Technologies, Inc.) stain or 100× CellMask stain (Thermo Fisher) for membrane staining. Cells were incubated in 96-well black-bottomed Greiner plates, and readings for relative fluorescence units (RFU) over 30 min of incubation were taken by a spectrophotometric plate reader (SpectraMax M5; Molecular Devices, CA) with excitation at 482 nm and emission at 504 nm for DiOC₆ and excitation at 485 nm and emission at 535 nm for CellMask green live stain.

Banerjee D., Umland T.C, & Panepinto J.C. (2016). De Novo Pyrimidine Biosynthesis Connects Cell Integrity to Amphotericin B Susceptibility in Cryptococcus neoformans. mSphere, 1(6), e00191-16.

+ Open protocol

+ Expand

Visualizing Cell Viability and Morphology

Check if the same lab product or an alternative is used in the 5 most similar protocols

The cells were visualized using confocal microscopy. First, the samples were fixed using frozen methanol and stained with DiOC-6 (Life Technologies, USA, CA) to visualize cell biological membranes, and propidium iodide (Sigma-Aldrich, Germany) to visualize cell nuclei. The cell area was determined using Ellipse software (Ellipse, Slovak Republic) from at least 3 independent images. Then, to visualize the viability of the cells, living cells were stained using BCECF-AM (Sigma-Aldrich, Germany) and propidium iodide to visualize dead cells. Viable cells metabolized nonfluorescent BCECF-AM to fluorescent BCECF (green color). propidium iodide does not penetrate into living cells, therefore only dead cell had red nuclei. The cells were counted using ImageJ software (NIH, USA) from at least 3 independent images, the viability of cells is expressed as percentage of viable cells of the total number of cells present on the scaffolds. The stained samples were observed using LSM 510 DUO confocal microscope (Zeiss, Germany). DiOC-6 (green color; maximum excitation wavelength 484 nm, maximum emission wavelength 501 nm), propidium iodide (red ORIGINAL RESEARCH color; maximum excitation wavelength 536 nm, maximum emission wavelength 617 nm), BCECF-AM (green color; maximum excitation wavelength 490 nm, maximum emission wavelength 535 nm).

Vocetková K., Sovková V., Buzgo M., Divín R., Amler E., & Filová E. (2020). FUNCTIONALIZATION OF POLYMERIC NANOFIBERS USING PLATELETS FOR MELANOCYTE CULTURE. Lékař a technika - Clinician and Technology, 50(1), 16-22.

+ Open protocol

+ Expand

Mitochondrial Membrane Potential Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Changes in MMP (∆ϕ_m) were examined with DiOC₆ (Molecular Probes, Eugene, OR, USA), as previously described [27 (link)]. Briefly, cells were treated with sterilized water or with holotoxin A₁ for the indicated times, then they were incubated with DiOC₆ (40 nM) for 20 min at 37 °C. Then, cells were washed and analyzed with flow cytometry. Finally, the percentage of cells with low MMP was calculated. For each sample, 10⁴ cells were investigated, and all experiments were performed in triplicate.

Yun S.H., Sim E.H., Han S.H., Han J.Y., Kim S.H., Silchenko A.S., Stonik V.A, & Park J.I. (2018). Holotoxin A1 Induces Apoptosis by Activating Acid Sphingomyelinase and Neutral Sphingomyelinase in K562 and Human Primary Leukemia Cells. Marine Drugs, 16(4), 123.

+ Open protocol

+ Expand

Mitochondrial Membrane Potential Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

DiOC₆ (Molecular Probes, Eugene, OR, USA) was added to the medium (1 µM final concentration) and incubated for 30 min. To investigate MMP, flow cytometry was performed using a CYTOMICS FC500 system (Beckman Coulter, Brea, CA, USA). The data were analyzed using CXP software version 2.2. The analyzer's threshold on the FSC channel was adjusted to exclude noise and most of the subcellular debris.

Yoon J.Y., Jeon H.O., Kim E.J., Kim C.H., Yoon J.U., Park B.S., Yu S.B, & Kwak J.W. (2017). Propofol protects human keratinocytes from oxidative stress via autophagy expression. Journal of Dental Anesthesia and Pain Medicine, 17(1), 21-28.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!