Fast dio

Manufactured by Thermo Fisher Scientific

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Fast-DiO is a fluorescent dye used for labeling cell membranes. It can be used to visualize cell morphology and dynamics in live-cell imaging applications.

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Fast DiO membrane dye (5 citations)

17 protocols using fast dio

Visualizing Amphid Neurons in P. pacificus

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DiO staining of amphid neurons was adapted from published staining of P. pacificus^{64 (link)}. Larval J2 P. pacificus were stained for 2 hours on a nutator in a solution of 15 ng/ml Fast DiO (ThermoFisher D3898) and then de-stained on an empty NGM plate for 1 hour. A 3% agar plug was used to gently transfer stained J2 animals onto a 2% agarose pad (melted in M9) with 20 mM sodium azide paralytic. Pairs of amphid neurons were ablated using an Andor Micropoint focused laser microbeam system. Cell identification was based on the identities described by Hong and colleagues^{65 (link)}. Cell death was confirmed by identifying a morphological change within the cell, and by re-staining after behavior was recorded. Each ablated J2 was transferred onto its own bacterial patch to recover before being used 2 days later to measure biting incentive.

Quach K.T, & Chalasani S.H. (2022). Flexible reprogramming of Pristionchus pacificus motivation for attacking Caenorhabditis elegans in predator-prey competition. Current biology : CB, 32(8), 1675-1688.e7.

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Measuring Phase Separation in Giant Plasma Membrane Vesicles

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Briefly, cells were grown to 60-80% confluence, then washed in GPMV buffer (10 mM HEPES, 150 mM NaCl, 2 mM CaCl₂, pH 7.4, in dH₂O). Cell membranes were then stained with 5 μg/mL of FAST-DiO (Thermo Fisher Scientific #D-3898), a lipidic dye that strongly partitions to L_d phases due to double bonds in its fatty anchors. Then, GPMVs were extracted by adding a solution of GPMV buffer + 25 mM PFA + 2 mM DTT and incubated at 37°C for 2 hours (HMLE) or 1 hour (T47D). The GPMV-containing solution was collected and mounted on BSA-coated coverslips for imaging.
The mounts were attached to a Peltier/temperature controlled microscope stage and imaged with green florescent laser under 40x objective. At each condition, 50-100 vesicles were counted as either phase separated or phase combined.
Increasing the temperature of the vesicles causes miscibility of the two phases. We raised the temperature in 3°C increments, at every increment counting each vesicle as either phase separated or phase combined (Figure 1A, Supplementary Figure 2). We continued these measurements until nearly all GPMVs were no longer microscopically phase separated. We then evaluated the change in temperature at which 50% of the GPMVs were phase separated by fitting to a sigmoid curve.

Tisza M.J., Zhao W., Fuentes J.S., Prijic S., Chen X., Levental I, & Chang J.T. (2016). Motility and stem cell properties induced by the epithelial-mesenchymal transition require destabilization of lipid rafts. Oncotarget, 7(32), 51553-51568.

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Multicolor Labeling of Cellular Components

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The following reagents were purchased from BioLegend: anti-Thy1 primary (1° Ab) antibody [mouse anti-human and mouse anti-rat, both unlabeled, fluorescein isothiocyanate (FITC)–labeled, and Alexa Fluor 647–labeled] and OKT3 (anti-CD3 antibody). ICAM-1 was purchased from Sino Biological. The following were purchased from Thermo Fisher Scientific: phospho-Erk1/2 (Tyr²⁰⁴), Fast-DiO, Fast-DiI, DiD, and Streptavidin–Alexa Fluor 488 or 647. 1,2-Distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE)-PEG-KK114 and 1,2-Dioleoyl-sn-glycero-3-phosphorylethanolamine DOPE-PEG-KK114 were a gift from E. Sezgin’s laboratory. DOPC, DPPC, cholesterol, trDHPE, LissRhod-DOPE, DO-NTA(Ni), and DP-NTA(Ni) were purchased from Avanti Polar Lipids. DSIDA was a gift from J. Stachowiak’s laboratory. Naphtho[2,3-a]pyrene was purchased from TCI America. Myr was purchased from Cayman Chemicals. ZA was purchased from Sigma-Aldrich.

Wang H.Y., Chan S.H., Dey S., Castello-Serrano I., Rosen M.K., Ditlev J.A., Levental K.R, & Levental I. (2023). Coupling of protein condensates to ordered lipid domains determines functional membrane organization. Science Advances, 9(17), eadf6205.

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Staining HeLa Cells for GPMV Formation

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HeLa cells were cultured at 37 °C and 5% CO₂ in Dulbecco’s modified eagle medium (DMEM, HyClone, USA) containing 10% fetal bovine serum (FBS, Biological Industries, USA), 100 units/ml antibiotic antimycostic solution (Sigma-Aldrich, USA), 1 mM sodium pyruvate (HyClone, USA), and 1.5 g/L sodium bicarbonate (HyClone, USA). Prior to the GPMV formation step, cell membranes were stained with 5 μg∕mL of ClO₄ (3,3′-Dilinoleyloxacarbocyanine Perchlorate) (Fast-DiO, Thermo Fisher Scientific, USA) for 10 min.

Chiang P.C., Tanady K., Huang L.T, & Chao L. (2017). Rupturing Giant Plasma Membrane Vesicles to Form Micron-sized Supported Cell Plasma Membranes with Native Transmembrane Proteins. Scientific Reports, 7, 15139.

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Lipid and Fluorophore Preparation for Membrane Studies

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Lipids POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine), C1P 18:1/d18:1 (N-oleoyl-ceramide-1-phosphate) and C1P 16:0/d18:1 (N-palmitoyl-ceramide-1-phosphate) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Fluorophores NBD-Cholesterol and TopFluor-C1P were purchased from Avanti Polar Lipids (Alabaster, AL, USA), fast DiO was purchased from ThermoFisher Scientific (Massachusetts, USA) and both Atto550-DMPE and Atto488 DOPE were purchased from Sigma Aldrich (Missouri, USA). Structures of lipids and ceramides are presented in Fig. S11. Doodle of fluorophores showing binding position is presented in Fig. S12. Sucrose and glucose were purchased from Sigma Aldrich (Missouri, USA). Low-melting temperature agarose polymer (

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C) was purchased from Fisher Scientific (Massachusetts, USA). The ultra-pure water used in experiments was obtained from a water purification system (Millipore).

Drabik D., Drab M., Penič S., Iglič A, & Czogalla A. (2023). Investigation of nano- and microdomains formed by ceramide 1 phosphate in lipid bilayers. Scientific Reports, 13, 18570.

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Measuring Membrane Phase Transitions

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Miscibility transition temperatures (T_misc) measurements were performed as previously reported ^{98 (link), 99 (link)}. Briefly, cells were washed in PBS, and cell membranes were labeled with 5 µg/ml fluorescent disordered/nonraft phase marker FAST DiO (Thermo Fisher Scientific) for 10 min on ice. Cells were then washed twice in GPMV buffer (10 mM HEPES, 150 mM NaCl, 2 mM CaCl2, pH 7.4), and then incubated with GPMV buffer supplemented with 25 mM paraformaldehyde (PFA) and 2 mM dithiothreitol (DTT) for 1 h at 37 °C. Vesicles were imaged at 40× on an inverted epifluorescence microscope (Leica DMi8) under temperature-controlled conditions using a microscope stage equipped with a Peltier element (Warner Instruments). GPMVs were imaged from 4° C-28 °C, counting phase-separated and uniform vesicles at each temperature. For each temperature, 25–50 vesicles were counted and the percent of phase-separated vesicles were calculated, plotted versus temperature, and a fitted to a sigmoidal curve to determine the temperature at which 50% of the vesicles were phase-separated (T_misc).

Henry W.S., Müller S., Yang J.S., Innes-Gold S., Das S., Reinhardt F., Sigmund K., Phadnis V.V., Wan Z., Eaton E., Sampaio J.L., Bell G.W., Viravalli A., Hammond P.T., Kamm R.D., Cohen A.E., Boehnke N., Hsu V.W., Levental K.R., Rodriguez R, & Weinberg R.A. (2024). Ether lipids influence cancer cell fate by modulating iron uptake. bioRxiv.

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Visualizing Drug Transport in Co-Culture

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Cells were pre-treated with 10 μg/ml doxorubicin (Dox; Pfizer, ADRIAMYCIN™, Bentley, Australia) or 2.5 μg/ml Fast-DiO (Thermo Fisher Scientific, D3898) for 30 min. After incubation, cells were washed with 1X PBS three times before the co-culture experiments. Then, treated cells were co-cultured with a 1:1 ratio with the untreated cells at the high-density condition for 4, 12, or 24 h. After the co-culture, cells were collected for flow cytometry analysis. The doxorubicin or Fast-DiO positive cells were gated and analyzed by FlowJo™. The median fluorescence intensity (MFI) ratio was calculated with the variation of the median fluorescence intensity of the treated cells to the untreated control cells. The time-lapse image of the drug transportation process was captured by the automated inverted microscope (Ti-Eclipse, Nikon, Tokyo, Japan) and the camera (ORCA-Flash4.0, Hamamatsu, Japan) under the onstage incubator for the general culture conditions.

Wu S.Y., Yu W.J., Chien T.Y., Ren Y.A., Chen C.S, & Chiang C.S. (2024). Microglia-mediated drug substance transfer promotes chemoresistance in brain tumors: insights from an in vitro co-culture model using GCV/Tk prodrug system. Cancer Cell International, 24, 35.

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Antibody Optimization and Drug Treatment Protocol

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All antibodies were diluted in TBS-T (Tris-buffered saline–Tween; 0.1%) treated with 3% bovine serum albumin (BSA) and stored at −20°C unless otherwise specified. Rabbit phospho-Akt (ser473) (D9E) (Cell Signaling Technology; 4060) was used at 1:1,000 for Western blotting (WB). Rabbit Akt antibody (Cell Signaling Technology; 9272) was used at 1:1,000 to detect total Akt in WB. Monoclonal anti-beta-actin antibody (Sigma-Aldrich; A1978) was used at 1:10,000 for WB. Mouse monoclonal US3 7H10.21 (21 (link)) was used at 1:1,000 for WB. Mouse monoclonal anti-Puromycin clone 4G11 (EMD Millipore; MABE342) was used at 1:2,000 for WB.
All drug treatments occurred 1 h prior to infection, concentrations represent final experimental concentrations, and drugs were resuspended in dimethyl sulfoxide (DMSO) unless otherwise specified. LY294002 (Cell Signaling Technology; 9901) was used at a concentration of 20 μM. Akt inhibitor VIII (Sigma; 124018) was used at a concentration of 5 μM. Rapamycin (Sigma; 553210) was used at a concentration of 100 μM. U0126 (Sigma; 662009) was used at a concentration of 10 μM. Puromycin (AG Scientific; P-1033-SOL) was solubilized in deionized H₂O and used at a concentration of 1 μg/mL. FAST-DiO (Thermo Fisher; D3898) was used a concentration of 5 μg/mL and incubated for 12 h prior to infection (24 h prior to imaging).

Esteves A.D., Koyuncu O.O, & Enquist L.W. (2022). A Pseudorabies Virus Serine/Threonine Kinase, US3, Promotes Retrograde Transport in Axons via Akt/mToRC1. Journal of Virology, 96(5), e01752-21.

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Fate Mapping of Otic Placode Development

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Fertilized chicken eggs (B&E farm, Maryland) were incubated at 39°C for various days and staged according to Hamburger and Hamilton (HH; 1992). Incubated eggs were windowed and injected with black India ink (Pelican) underneath the embryo to enhance contrast. For fate mapping study, at HH St13 (19-20 somite stage (ss), Embryonic day 2 (E2)), lipophilic tracers, CM-DiI or FAST DiO (Molecular Probes, # C-7000 and D-3898), was injected at designated locations on the rim of the otic cup according to a clock face grid (Fig. 2; (Brigande et al., 2000 (link))). Working solution for both dyes was prepared by 1:10 dilution of CM-DiI (1mg/ml) or DiO (2mg/ml) stock solution prepared in 70% dimethylformamide.
For transplantation surgeries, an E2 donor embryo was transferred to a Sylgard dish. The left otic cup was injected with dyes and isolated using a tungsten needle and a homemade microblade. Then, the right otic cup of an age-matched host embryo was removed and replaced with a donor's left otic cup aligned to the same anterior-posterior axis as the host. Digital photographs were taken before, during, and after the surgery for documentation. After surgery, Tyrode's solution was applied to the embryo before sealing the egg and returning to the incubator until harvest.

Deng X, & Wu D.K. (2016). Temporal coupling between specifications of neuronal and macular fates of the inner ear. Developmental biology, 414(1), 21-33.

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GUV Electroformation and Confocal Imaging

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Chamber for GUV electroformation consisted of two conductive ITO glasses separated by a rubber spacer of 0.3 mm. Phospholipid extracts were dissolved in chloroform/methanol (2:1) and deposited on one of the ITO coated glasses. After drying the chamber was filled with 300 μl of 0.1 M Tris-HCl pH 7.5 and 200 mM sucrose. AC field of 10 Hz, 1 V was applied to the chamber. Electroformation was conducted at 40°C for 2 hours [89 (link)].
FAST DIO (Molecular Probes) was added at a concentration of 0.1 mol% to GUV mixtures followed by spinning disk confocal imaging at excitation 488 nm. Images were acquired with the same settings and intensities of the individual GUVs were analyzed at the equatorial plane.

Makarova M., Peter M., Balogh G., Glatz A., MacRae J.I., Lopez Mora N., Booth P., Makeyev E., Vigh L, & Oliferenko S. (2020). Delineating the Rules for Structural Adaptation of Membrane-Associated Proteins to Evolutionary Changes in Membrane Lipidome. Current Biology, 30(3), 367-380.e8.

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