Atto647n dppe

Manufactured by ATTO-TEC

Sourced in Germany

Atto647N-DPPE is a fluorescent dye molecule used as a labeling agent for biomolecules. It is a synthetic, organic compound that emits light in the far-red region of the visible spectrum when excited by appropriate wavelengths of light.

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Lab products found in correlation

L glutamine, by Merck Group (2 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Merck Group (2 mentions) Lsm 780, by Zeiss (2 mentions) Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions) E coli polar lipid extract, by Avanti Polar Lipids (1 mentions) Atto488 dppe, by ATTO-TEC (1 mentions) Atto 565 dppe, by ATTO-TEC (1 mentions) L 15 medium, by Thermo Fisher Scientific (1 mentions) Hepes ph 7, by Merck Group (1 mentions) 8 well glass bottom chambers, by Ibidi (1 mentions) Rpmi 1640, by Merck Group (1 mentions) Penicillin, by Merck Group (1 mentions) Streptomycin, by Merck Group (1 mentions) Streptavidin, by Thermo Fisher Scientific (1 mentions) Biotinylated bsa, by Merck Group (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) 1 2 distearoyl sn glycero 3 phosphoethanolamine n biotinyl polyethylene glycol 2000 dspe peg biotin, by Avanti Polar Lipids (1 mentions) Brain pi 4 5 p2, by Avanti Polar Lipids (1 mentions) Brain l α ps, by Avanti Polar Lipids (1 mentions)

7 protocols using atto647n dppe

Ptk2 Cells for Membrane Diffusion Analysis

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Potorous
tridactylus epithelial kidney (Ptk2) cells were cultured in DMEM (Sigma-Aldrich,
UK) supplemented with 15% fetal bovine serum (Sigma-Aldrich) and 1% l-glutamine (Sigma-Aldrich).
For microscopy experiments,
the cells were seeded onto 25 mm glass coverslips and allowed to grow
to a confluence of about 75%. For sFCS experiments, the cells were
labeled with Atto647N-DPPE or Atto647N-sphingomyelin (AttoTec, Germany)
by incubating the cells at a lipid analogue concentration of 0.4 μg/μL
in L15 (Sigma-Aldrich) at room temperature for 15 min. After cells
were washed twice with L15, the experiments were performed at 37 °C.
The diffusion of the lipid analogues in the cellular membrane was
measured in the periphery of the cell at its bottom membrane.
Transfections of Ptk2 cells with GPI-anchored green fluorescent
protein were performed using Lipofectamine 3000 (Thermo Fisher) according
to the manufacturer’s protocol. The GPI-GFP plasmid was obtained
from Kai Simons’ Lab (Max Planck Institute of Molecular Cell
Biology and Genetics, Dresden, Germany).

Schneider F., Waithe D., Lagerholm B.C., Shrestha D., Sezgin E., Eggeling C, & Fritzsche M. (2018). Statistical Analysis of Scanning Fluorescence Correlation Spectroscopy Data Differentiates Free from Hindered Diffusion. ACS Nano, 12(8), 8540-8546.

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Preparation of Bacterial Lipid Stocks

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To prepare the lipid stock solution, E. coli polar lipid extract (Avanti Polar Lipids, Inc.) was dissolved in water and sonicated for 30 min to make a 30-mg/mL aqueous suspension stock. The lipid stock solution was flash frozen in liquid nitrogen and stored at −80°C. To prepare a 2 mg/mL aqueous suspension stock of lipopolysaccharide, LPS from E. coli EH100 (Ra mutant; Sigma) was dissolved in water and sonicated for 30 min. The lipopolysaccharide stock solution was flash frozen in liquid nitrogen and stored in aliquots at −80°C.
To prepare the fluorescent lipid stock solutions for flow cytometry, E. coli polar lipid extract (Avanti Polar Lipids, Inc.) was dissolved in chloroform with 1% (molar ratio) ATTO-488 DPPE, ATTO-565 DPPE, or ATTO-647N DPPE (ATTO-TEC GmbH). Lipid mixtures were then dried into a film, hydrated, and lyophilized overnight. The lipid mixtures were then dissolved in water and sonicated for 30 min to make a 30-mg/mL aqueous suspension stock. The lipid stock solution was flash frozen in liquid nitrogen, and stored at −80°C.

Falchi F.A., Taylor R.J., Rowe S.J., Moura E.C., Baeta T., Laguri C., Simorre J.P., Kahne D.E., Polissi A, & Sperandeo P. (2022). Suppressor Mutations in LptF Bypass Essentiality of LptC by Forming a Six-Protein Transenvelope Bridge That Efficiently Transports Lipopolysaccharide. mBio, 14(1), e02202-22.

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Jurkat T-cell Labeling and Measurement

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Jurkat T-cells were cultured in RPMI-1640 (Sigma Aldrich, UK) media supplemented with 10% FBS (Sigma Aldrich), 2 mM l-Glutamine (Sigma Aldrich), 100 U/mL Penicillin (Sigma Aldrich), 0.1 mg/mL Streptomycin (Sigma Aldrich) and 10 mM HEPES pH 7.4 (Sigma Aldrich). 1 million cells were spun down for 5 min at 2000 rpm and washed with 1 mL of L15 medium (Life Technologies). After spinning down again the cells were labelled by resuspending in L15 medium containing 0.4 μg/mL Atto647N-DPPE (AttoTec). The cells were labelled at 37 °C shaking at 300 rpm for 15 min. After washing with L15, the cells were resuspended and kept in L15 for not longer than 1 h on room temperature. Measurements were performed in 8-well glass-bottom chambers (Ibidi). Prior to the measurements the glass was coated with PLL using a 0.01% PLL-solution (Poly-l-Lysine) (Sigma Aldrich) for 1 h at room temperature and washed three times with L15.

Waithe D., Schneider F., Chojnacki J., Clausen M.P., Shrestha D., de la Serna J.B, & Eggeling C. (2018). Optimized processing and analysis of conventional confocal microscopy generated scanning FCS data. Methods (San Diego, Calif.), 140-141, 62-73.

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Phase Separation in GPMVs Imaged

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For experiments with phase-separated GPMVs, the membranes were labelled with a fluorescent lipid analogue Atto647N-DPPE (AttoTec) by incubating the GPMV solution with the dye at up to 0.02 g/ml for 5-10 min, followed by a 20-min incubation with up to mM deoxycholic acid (DCA) to facilitate large-scale phase separation (46) . A drop of the GPMV suspension was then transferred into the chamber created with Vaseline between two coverslips (22  50 mm, thickness no. 1.5; by Menzel), which was adhered to a Peltier cooling device set to 4 °C mounted on the microscope stage (35) .
The observations were conducted using an inverted laser-scanning confocal microscope Zeiss LSM780 equipped with a 40x/1.1 water-immersion objective. Green and far-red fluorescence was excited with lasers at 488 nm and 633 nm and collected in two channels within the spectral windows of 500-590 nm and 640-700 nm. For each vesicle, a z-stack of images across the entire vesicle was collected with spacing between slices of 0.56 m and sampling of around 0.1 m/pixel. Acquisition of each slice took 2-6 seconds, which was fast enough that the locations of protein aggregates were not smeared by their diffusion.

Urbančič I., Schiffelers L.D.J., Jenkins E., Gong W., Santos A.M., Schneider F., O’Brien‐Ball C., Vuong M.T., Ashman N., Sezgin E., & Eggeling C. (2020). Aggregation and immobilisation of membrane proteins interplay with local lipid order in the plasma membrane of T cells.

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Lipid Membrane Composition Analysis

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Lipids 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol (Chol), egg sphingomyelin (SM), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG-biotin) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). The fluorescent lipid analogues Atto647N-DPPE and C-Laurdan were purchased from Atto-Tec GmbH (Siegen, Germany) and 2pprobes (Seoul, Korea), respectively. Bovine serum albumin (BSA) and biotinylated BSA were obtained from Sigma Aldrich (Gillingham, UK), whereas streptavidin was provided by ThermoFisher (Waltham, MA, USA).

Urbančič I., Brun J., Shrestha D., Waithe D., Eggeling C, & Chojnacki J. (2018). Lipid Composition but Not Curvature Is the Determinant Factor for the Low Molecular Mobility Observed on the Membrane of Virus-Like Vesicles. Viruses, 10(8), 415.

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Lipid Analogs for Fluorescence Microscopy

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Lipids 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), brain l-α-PS, and brain PI(4,5)P2 were purchased from Avanti Polar Lipids (Alabaster, AL, USA). ATTO647N-DPPE and ATTO647N–SM (ATTO647N-SM) were purchased from ATTO-TEC GmbH (Siegen-Weidenau, Siegen, Germany). Abberior STAR RED (KK114)–1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (KK114-DPPE), cholesterol–PEG(1 k)-Abberior STAR RED (KK114) [Chol-PEG(1 k)-KK114], and ATTO647N-PI(4,5)P2 [ATTO647N-PI(4,5)P2] were all purchased from Abberior GmbH (Göttingen, Germany). Chemical structures of all the lipid analogs used in this study are given in fig. S1.

Favard C., Chojnacki J., Merida P., Yandrapalli N., Mak J., Eggeling C, & Muriaux D. (2019). HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly. Science Advances, 5(10), eaaw8651.

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Supported Lipid Bilayer Preparation

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For tracking, a solution consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (Avanti): CHOL (Avanti) and Abberior STAR 488-CHOL (50:49.5:0.5), doped with ATTO 647N-DPPE (ATTO-TEC GmbH) was dissolved in 50/50 chloroform/ethanol. Supported lipid bilayers were created by spin coating (3000 rpm for 30 s) this solution onto coverslips. To keep the layer hydrated, the coverslip was placed in a sample chamber and covered with PBS.

Schmidt R., Weihs T., Wurm C.A., Jansen I., Rehman J., Sahl S.J, & Hell S.W. (2021). MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope. Nature Communications, 12, 1478.

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