Rhodamine dextran

Manufactured by Merck Group

Sourced in United States

Rhodamine-dextran is a fluorescent dye conjugated to dextran, a polysaccharide. It can be used as a tracer or labeling agent in biological research applications.

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28 protocols using rhodamine dextran

Endothelial Permeability Assessment Using Exosomes

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In vitro endothelial permeability was assessed by quantifying the amount of rhodamine B isothiocyanate-dextran (rhodamine-dextran, average MW ~70,000; Sigma-Aldrich) that passed through the endothelial monolayers. Briefly, endothelial cells (2 × 10⁴) were plated on a 24-well Transwell filter (0.4-μm pore size; Corning), and 10% exosome-depleted FBS-1640 medium with exosomes derived from NPC cells with or without EBV at the concentration of 5 μg ml⁻¹ was added into both upper and lower chambers, and then incubated for 48 h to allow the cells to reach 100% confluence. rhodamine-dextran was then added to the upper chamber at 20 mg ml⁻¹, and 1 h later, the amount of rhodamine-dextran in the lower chamber was determined by measuring the fluorescence intensity using a microplate spectrofluorometer (BioTek, Vermont, USA) at a 544 nm excitation and a 590 nm emission wavelength. The investigator was blinded to the group allocation.

Li D.K., Chen X.R., Wang L.N., Wang J.H., Li J.K., Zhou Z.Y., Li X., Cai L.B., Zhong S.S., Zhang J.J., Zeng Y.M., Zhang Q.B., Fu X.Y., Lyu X.M., Li M.Y., Huang Z.X, & Yao K.T. (2022). Exosomal HMGA2 protein from EBV-positive NPC cells destroys vascular endothelial barriers and induces endothelial-to-mesenchymal transition to promote metastasis. Cancer Gene Therapy, 29(10), 1439-1451.

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In Vivo Imaging of Tumor Angiogenesis

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To observe U87-miR-378 and U87-GFP tumor growth and angiogenesis in the brain of mice under CWs, multi-photon laser scanning microscopy (MPLSM) imaging was used. It was conducted on a custom-built MPLSM using a confocal laser-scanning microscope body (BX-51, Optical Analysis) and a femtosecond laser source (High Performance Mai Tai, Spectra-Physics) [21 (link), 22 (link), 38 (link)]. Imaging studies were performed at 20 magnification, 0.95 NA water immersion objective (Olympus XLUMPlanFl, 1-UB965, Optical Analysis). Prior to each imaging session, mice were anesthetized by intraperitoneal injection of Xylazine/Ketamine (10 and 1.0 mg/kg, respectively). The tail vein was injected with 100 μl rhodamine–dextran (Sigma-Aldrich) and positioned on a stereotactic frame. For each tumor, four adjacent images were obtained through the CW at day 3, and the same regions were revisited and recorded at day 7 post-implantation. The percentage of tumor vessels was analyzed by Image J software (http://rsbweb.nih.gov/ij/download.html).

Li W., Liu Y., Yang W., Han X., Li S., Liu H., Gerweck L.E., Fukumura D., Loeffler J.S., Yang B.B., Jain R.K, & Huang P. (2017). MicroRNA-378 enhances radiation response in ectopic and orthotopic implantation models of glioblastoma. Journal of neuro-oncology, 136(1), 63-71.

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Reagents and Solutions for Cell Culture

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The following reagents and solutions were used. Agarose (BP1356-100; Thermo Fischer Scientific), BSA (A4503; Sigma-Aldrich), fibronectin (F1141; Sigma-Aldrich), FDx (D1821; Sigma-Aldrich), gelatin (G1890; Sigma-Aldrich), methylcellulose (M0387-100; Sigma-Aldrich), Mowiol 40-88 (81386; Fluka), penicillin-streptomycin (P4458; Sigma-Aldrich), Rhodamine-dextran (D1824; Sigma-Aldrich), sucrose and (27480.294; VWR). Danilchick’s medium 1×: 53 mM NaCl, 5 mM NA₂CO₃, 4.5 mM potassium gluconate, 32 mM sodium gluconate, 1 mM MgSO₄-7H₂0, 1 mM CaCl₂, and 0.1% BSA. MEMFA: 1 mM MOPS, 2 mM EGTA, 1 mM MgSO₄, and 3.7% formaldehyde. Normal amphibian medium (NAM): 110 mM NaCl, 2 mM KCl, 1 mM Ca(CO₃)₂, 1 mM MgSO₄, 0.1 mM EDTA, 1 mM NaHCO₃, and 2 mM sodium phosphate. NTMT: 0.1 M NaCl, 0.1 M TrisHCl, pH 9.5, 50 mM MgCl₂, and Tween 0.1%. PBS 1×: 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 1 mM CaCl₂-2H₂O, and 0.5 mM MgCl₂-6H₂O.

Kuriyama S., Theveneau E., Benedetto A., Parsons M., Tanaka M., Charras G., Kabla A, & Mayor R. (2014). In vivo collective cell migration requires an LPAR2-dependent increase in tissue fluidity. The Journal of Cell Biology, 206(1), 113-127.

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Targeting tgs1 in Zebrafish Embryos

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Control (non-targeting) and tgs1 morpholino oligonucleotides (MOs) were purchased from (GeneTools, LLC) using the tgs1 XM_003197865.5 sequence as reference. MO sequences are reported in Supplementary Table S11. For TGS1 mRNA injection, human TGS1 cDNA (NM_001317902) was cloned into an N-terminal flag-pCS2 + mRNA expression vector. In vitro transcription of 5′-capped mRNAs was performed using the mMACHINE SP6 Transcription Kit (Ambion) following the manufacturer's protocol and as previously described in (33 (link),34 (link)). Embryos from TL/EK wild-type crossings were used to visualize the CaP-MN phenotype. Embryos were injected with the respective dose of MOs or mRNA in an aqueous solution containing 0.05% PhenolRed and 0.05% Rhodamine-Dextran (Sigma-Aldrich). At 6–7 h after injection, embryos were sorted according to homogeneity of the rhodamine fluorescence signal.

Chen L., Roake C.M., Maccallini P., Bavasso F., Dehghannasiri R., Santonicola P., Mendoza-Ferreira N., Scatolini L., Rizzuti L., Esposito A., Gallotta I., Francia S., Cacchione S., Galati A., Palumbo V., Kobin M.A., Tartaglia G.G., Colantoni A., Proietti G., Wu Y., Hammerschmidt M., De Pittà C., Sales G., Salzman J., Pellizzoni L., Wirth B., Di Schiavi E., Gatti M., Artandi S.E, & Raffa G.D. (2022). TGS1 impacts snRNA 3′-end processing, ameliorates survival motor neuron-dependent neurological phenotypes in vivo and prevents neurodegeneration. Nucleic Acids Research, 50(21), 12400-12424.

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Endothelial Cell Permeability Assay

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For permeability assays, endothelial cells were cultured on collagen gels as described above. Monolayers were washed with PBS and 100 µg/ml 10-kD rhodamine-Dextran (Sigma-Aldrich) was added to each well. Monolayers were then incubated at 37°C in 5% CO₂ for 1 h. After 1 h, monolayers were then washed extensively with PBS and the relative fluorescence intensity of rhodamine-Dextran that had passed across the monolayers into the collagen gels was measured by a FilterMax F5 microplate reader (Molecular Devices). Indicated results are the mean fluorescence intensity of at least three independent experiments with at least replicates for each sample.

Sullivan D.P., Dalal P.J., Jaulin F., Sacks D.B., Kreitzer G, & Muller W.A. (2019). Endothelial IQGAP1 regulates leukocyte transmigration by directing the LBRC to the site of diapedesis. The Journal of Experimental Medicine, 216(11), 2582-2601.

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Biopolymer-Based Hydrogel Fabrication

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Sodium alginate was purchased from Pronova Biopolymers (Oslo, Norway) with an average molecular weight of ~250 kDa and with high guluronate content (Protoanal LF 20/40). Adipic acid dihydrazide (AAD), 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC), MES, 1-hydroxybenzotriazole (HOBT), iron (II,III) oxide powder (< 5 micron), mitoxantrone, phosphate buffered saline (PBS), bovine serum albumin (BSA), Sigmacote, Fluorescein isothiocyanate (FITC)-dextrans, Rhodamine-dextran, and FITC-Diethylaminoethyl (DEAE)-dextrans were all purchased from Sigma-Aldrich (St. Louis, MO). Mouse Bone Morphogenetic Protein-2 (BMP-2), Thymus Chemokine-1 (TCK-1), and Enzyme-linked Immunosorbent Assay (ELISA) kits and kit reagents were purchased from R&D Systems (Minneapolis, MN).

Kennedy S., Roco C., Déléris A., Spoerri P., Cezar C., Weaver J., Vandenburgh H, & Mooney D. (2018). Improved magnetic regulation of delivery profiles from ferrogels. Biomaterials, 161, 179-189.

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Lysozyme-Dextran Nanogel Synthesis

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Lysozyme-dextran nanogels (LDNGs) were synthesized as previously described (Li et al., 2008 (link); Coll Ferrer et al., 2014 (link); Myerson et al., 2018 (link), 2022 (link)). Rhodamine-dextran or FITC-dextran (Sigma) and lysozyme from hen egg white (Sigma) were dissolved in deionized and filtered water at a 1:1 or 2:1 mol:mol ratio. Then pH was adjusted to 7.1 and solution was lyophilized. For Maillard reaction, the lyophilized product was heated for 18 h at 60°C, with 80% humidity maintained via saturated KBr solution in the heating vessel. Dextran-lysozyme conjugates were dissolved in deionized and filtered water to a concentration of 5 mg/ml. Solutions were stirred at 80°C for 30 min. Diameter of LDNGs was evaluated with dynamic light scattering (DLS, Malvern) after heat gelation. Particle suspensions were stored at 4°C.

Rubey K.M., Mukhitov A.R., Nong J., Wu J., Krymskaya V.P., Myerson J.W., Worthen G.S, & Brenner J.S. (2022). Nanoparticle-Induced Augmentation of Neutrophils’ Phagocytosis of Bacteria. Frontiers in Pharmacology, 13, 923814.

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Cell Transplantation in Zebrafish Embryos

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MZptk7, MZvangl2 or Xdd1mRNA-injected cells were labelled with rhodamine dextran (red 10,000 MW, Sigma) by injections at the one-cell stage. Labelled cells were transplanted from sphere (4 hpf) stage embryos into the ventral margin of unlabelled WT hosts at shield stage (6 hpf) as previously described38 (link). Embryos were screened at 24 hpf on an Axio Imager.M1 (Zeiss) compound microscope and embryos containing significant rhodamine-labelled cells (mutant/Xdd1 cells) in the embryonic tail were grown to larval stages.

Hayes M., Gao X., Yu L.X., Paria N., Henkelman R.M., Wise C.A, & Ciruna B. (2014). ptk7 mutant zebrafish models of congenital and idiopathic scoliosis implicate dysregulated Wnt signalling in disease. Nature Communications, 5, 4777.

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Measuring Gap Junction Functionality

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This procedure was performed according to the method described in an earlier report [40 (link)]. Briefly, liver samples were obtained from Wt rats with each treatment (4 rats per group) and treated with Lucifer yellow (Sigma-Aldrich Corp., St. Louis, MO), a stain that can pass through the gap junction channel, and rhodamine-dextran (Sigma-Aldrich Corp.), which does not cross through the channel, to measure gap junction capability. Liver slices were cut to 5 mm-thick and 3 incisions of 1 mm depth were made, followed by the addition of a mixture of fluorescent dyes containing 0.05% Lucifer yellow and 0.05% rhodamine-dextran in PBS into the incisions. After 3 minutes, the slices were washed 3 times with PBS and frozen. Thereafter 7 μm thick frozen sections were made and spread of the dye was measured using an image analyzer (Keyence).

Kato H., Naiki-Ito A., Naiki T., Suzuki S., Yamashita Y., Sato S., Sagawa H., Kato A., Kuno T, & Takahashi S. (2015). Connexin 32 dysfunction promotes ethanol-related hepatocarcinogenesis via activation of Dusp1-Erk axis. Oncotarget, 7(2), 2009-2021.

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Intravital Multiphoton Imaging of Bone Marrow

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Mice were prepared for intravital TPLSM as previously described^{4 (link)}. TPLSM was performed with a Leica system as described above. EGFP⁺ cells of 7-12 week old female LysM-EGFP mice and tdTomato⁺ cells of Catchup^IVM-red mice were excited at 960nm, at which bone tissue additionally emits a second-harmonic generation (SHG) signal at 480nm. Fluorescent cells were detected with specific filters at 525/50nm (EGFP) or 585/50nm (tdTomato) and SHG was detected via a 460/50nm filter.
Blood flow was visualized by injecting 1.5mg/ml Rhodamine Dextran (Sigma-Aldrich, Cat# R9379-100MG) or 1μM Qtracker™ 655 Vascular Labels (Thermo Fisher, Cat# Q21021MP) in a total volume of 100μl PBS i.v.. Fluorescence was excited at 960nm and detected with a 585/40nm (Rhodamine Dextran) or a 650/50nm (Qtracker 655) filter. Imaging was performed as well in resonant as in non-resonant detection mode. Scan speed was adjusted individually for different vessel types from 600 Hz to 12 KHz.
Neutrophils were activated by injecting 100μg/kg body weight hG-CSF (Neupogen®, Amgen GmbH) i.v. in a total volume of 100μl PBS. The raw data were reconstructed and analyzed using Imaris software (Bitplane) and ImageJ.
Further information on software versions used for data collection and processing are listed in the reporting summary document and Supplementary table 4.

Grüneboom A., Hawwari I., Weidner D., Culemann S., Müller S., Henneberg S., Brenzel A., Merz S., Bornemann L., Zec K., Wuelling M., Kling L., Hasenberg M., Voortmann S., Lang S., Baum W., Ohs A., Kraff O., Quick H.H., Jäger M., Landgraeber S., Dudda M., Danuser R., Stein J.V., Rohde M., Gelse K., Garbe A.I., Adamczyk A., Westendorf A.M., Hoffmann D., Christiansen S., Engel D.R., Vortkamp A., Krönke G., Herrmann M., Kamradt T., Schett G., Hasenberg A, & Gunzer M. (2019). A network of trans-cortical capillaries as mainstay for blood circulation in long bones. Nature metabolism, 1(2), 236-250.

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