Fitc cholera toxin b subunit

Manufactured by Merck Group

Sourced in United Kingdom, United States

FITC-cholera toxin B subunit is a fluorescently labeled protein used as a tool in biological research. It consists of the non-toxic B subunit of the cholera toxin molecule conjugated to the fluorescent dye fluorescein isothiocyanate (FITC). This complex binds to ganglioside GM1, a cell surface receptor, and is commonly used to label and visualize GM1-expressing cells and tissues.

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Cholera toxin (1100 citations) Cholera toxin B (9 citations) FITC-conjugated cholera toxin B subunit (5 citations) Cholera toxin B-FITC (4 citations) FITC-conjugated Cholera toxin B subunit (CTxB) (2 citations)

5 protocols using fitc cholera toxin b subunit

Neuronal Cell Imaging Assays

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Fetal calf serum, Dulbecco’s Minimal Essential Medium (DMEM) low-glucose medium and Roswell Park Memorial Institute (RPMI) medium were from Gibco (UK). Anti-EEA1 and anti-GRIN1 antibody were from Abcam (UK). Secondary antibodies were from Jackson Immunoresearch (USA). AlexaFluor555-conjugated Transferrin was from Thermo Fisher (UK). APV, MK801, memantine were from Tocris (UK). CdCl₂, CaCl₂ and FITC-cholera toxin B subunit were from Sigma-Aldrich (UK). Fluoromount-G mounting medium was from Southern Biotech (USA).

, & Glebov O.O. (2020). Tonic NMDA receptor signalling shapes endosomal organisation in mammalian cells. Scientific Reports, 10, 9315.

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DENV-specific Monoclonal Antibody Characterization

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The mouse anti-DENV monoclonal Ab 3H5 produced by the hybridoma HB46 (ATCC, USA), kindly supplied by Dr. Irene Bosch, University of Massachusetts Medical School, USA, and 2H2 (Chemicon, USA) were used. 3H5 is an IgG1 Ab that reacts with DENV-2 E protein and binds to IgG Fc receptor II (FcγRII) whereas 2H2 is an IgG2a Ab reactive with the prM protein of all members of DENV complex and binds to both FcγRI and FcγRII [26 (link)]. The clone AT10 of an anti-human FcγRII Ab was provided by Dr. Mirta Giordano (Academia Nacional de Medicina, Buenos Aires, Argentina).
TRITC-human transferrin was from Molecular Probes (USA) and FITC-cholera toxin B subunit was purchased from Sigma-Aldrich (USA). Chlorpromazine, dansylcadaverine, ammonium chloride, β-methylcyclodextrin, dynasore, and acridine orange were purchased from Sigma-Aldrich (USA).

Carro A.C., Piccini L.E, & Damonte E.B. (2018). Blockade of dengue virus entry into myeloid cells by endocytic inhibitors in the presence or absence of antibodies. PLoS Neglected Tropical Diseases, 12(8), e0006685.

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Fluorescent Labeling of Cells

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Cells (5 × 10⁵) were washed and resuspended in RPMI with 1% fetal bovine serum at 4 °C for 20 min. Ten microliters of Fluor-conjugated antibodies and, for some experiments, FITC-cholera toxin B subunit (Sigma) were then added for an additional 30 min. Cells were then washed and resuspended in 50 μl RPMI. Cells were incubated at 37 °C for the indicated times then fixed with ice-cold 4% paraformaldehyde for 20 min. Cells were washed and placed on poly-d-lysine-coated slides (Fisher Scientific, Pittsburgh, PA, USA). Coverslips were mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Inc. Bulingame, CA, USA). Cells were visualized using a Zeiss fluorescent Axioskop 2 microscope with a × 40 1.3 numerical aperture objective and Axiocam camera (Thornwood, NY, USA).

Jack J., Small G., Brown C., Havener T., McLeod H., Motsinger-Reif A, & Richards K. (2017). Gene expression and linkage analysis implicate CBLB as a mediator of rituximab resistance. The pharmacogenomics journal, 18(3), 467-473.

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Immunofluorescence Assay for Lipid Raft Visualization

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For immunofluorescence assays, cells were immobilized in PolyPrep slides (Sigma-Aldrich) for 15 min and then fixed with 2% paraformaldehyde (PFA)–0.025% glutaraldehyde in 1× PBS for 10 min at room temperature. After washing twice with 0.1% glycine/PBS, cells were permeabilized with 0.1% Triton X-100/PBS. Incubation with each primary and secondary antibodies and subsequent washes were performed with 1× PBS–2% BSA–0.05% saponine buffer. 4′,6-diamidino-2-phenylindole (Dapi) was used for nuclear staining while cholera toxin B subunit FITC (fluorescein isothiocyanate) conjugate was used as marker of membrane lipid rafts (Sigma-Aldrich). Images were obtained with Leica TCS-SP confocal microscope or Leica DMI 4000B Inverted Microscope (Leica Microsystems, Wetzlar, Germany). The intensity mean per pixel was calculated in confocal images using LAS AF Lite software (Leica Microsystems), and values were represented in bar diagrams showing statistical significance.

López-Huertas M.R., Li J., Zafar A., Rodríguez-Mora S., García-Domínguez C., Mateos E., Alcamí J., Rao S, & Coiras M. (2016). PKCθ and HIV-1 Transcriptional Regulator Tat Co-exist at the LTR Promoter in CD4+ T Cells. Frontiers in Immunology, 7, 69.

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Endocytosis of APRIL by Astrocytoma Cells

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Human astrocytes were purchased from ScienCell Research Laboratories (Carlsbad, CA). The astrocytoma cell line CRT was purchased from American Tissue Culture Collection (Manassas, VA). Primary mixed glial cultures were established from the forebrains of C57Bl/6J newborn mice. Amoeboid microglia floating cells were detached from the astroglial monolayer by manual shaking. The remaining adherent monolayer contained astrocytes and adherent microglia. The cells were detached with trypsin. The microglia were removed by adhesion on plastic over a period of 30 minutes. Lipopolysaccharide (LPS) and Poly-IC were from Sigma. For the endocytosis experiment, the CRT cell line was incubated with 1 μg/ml of human FLAG-tagged APRIL for 30 minutes at 4 C, washed, and further incubated at either 4 C or 37 C for 45 minutes. Cells were then fixed in paraformaldehyde 4% followed by quenching in phosphate-buffered saline (PBS) glycine 0.1 M. Astrocyte-bound APRIL was detected with a biotinylated anti-FLAG antibody (1 μg/ml, Sigma) and PEconjugated streptavidin (BD Biosciences) in PBS 2% bovine serum albumin 0.2% saponin. Cholera toxin B subunit FITC (10 μg/ml, Sigma) and DAPI (5 μg/ml) were used to label plasma membrane and nucleus, respectively. Cells were analyzed on an LSM510 confocal microscope (Carl Zeiss) with a plan-Apochromat ×63/1.4 oil objective.

Baert L., Benkhoucha M., Popa N., Ahmed M.C., Manfroi B., Boutonnat J., Sturm N., Raguenez G., Tessier M., Casez O., Marignier R., Ahmadi M., Broisat A., Ghezzi C., Rivat C., Sonrier C., Hahne M., Baeten D., Vives R.R., Lortat-Jacob H., Marche P.N., Schneider P., Lassmann H.P., Boucraut J., Lalive P.H, & Huard B. (2019). A proliferation-inducing ligand-mediated anti-inflammatory response of astrocytes in multiple sclerosis. Annals of neurology, 85(3).

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