Brefeldin a

Manufactured by Illumina

Sourced in United Kingdom

Brefeldin A is a fungal metabolite that inhibits the transport of proteins from the endoplasmic reticulum to the Golgi apparatus. It is commonly used as a research tool to study intracellular protein trafficking and secretion processes.

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Lab products found in correlation

Cycloheximide (chx), by Merck Group (4 mentions) Nocodazole, by Merck Group (3 mentions) Mouse anti cd63 mab, by Developmental Studies Hybridoma Bank (2 mentions) Mouse anti cd8a mab, by Developmental Studies Hybridoma Bank (2 mentions) Mouse anti eea1 mab, by BD (2 mentions) Mouse anti gm130 mab, by BD (2 mentions) Alexa fluor conjugated goat antibodies against mouse igg, by Thermo Fisher Scientific (2 mentions) Galnac o bn, by Merck Group (2 mentions) Mouse anti gfp mab, by Santa Cruz Biotechnology (2 mentions) Pngase f, by New England Biolabs (2 mentions) Chemerin, by R&D Systems (1 mentions) Igf 1, by R&D Systems (1 mentions) Ionomycin, by Merck Group (1 mentions) Rapamycin, by Merck Group (1 mentions) R59949, by Merck Group (1 mentions) Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions) Fibronectin antibody, by Merck Group (1 mentions) Ag1478, by Cell Signaling Technology (1 mentions) Phorbol myristate acetate (pma), by Merck Group (1 mentions) Tunicamycin, by Merck Group (1 mentions)

8 protocols using brefeldin a

Chemerin and Growth Factor Stimulation

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Cells (10⁶) were plated in 10cm dishes, incubated overnight, then washed 3 times with 10ml sterile PBS, and incubated in 5ml serum-free media (SFM) for 1 h followed, unless otherwise stated, by stimulation for 30 min with 100ng.ml^-1 chemerin (R&D Systems Inc., Oxfordshire, UK), 100ng.ml^-1 recombinant human GF-II, 50ng.ml^-1 IGF-I (R&D Systems Inc.) or 1μM ionomycin (Sigma-Aldrich, Poole, UK). In some experiments cells were preincubated for 30 min with 10μg.ml^-1 brefeldin A (Epicentre Biotechnologies, Cambio Ltd, Cambridge, UK), 10μg.ml^-1 cycloheximide (Sigma-Aldrich), 3.2μM AG1024 (Calbiochem) or 1μM CCX832 (ChemoCentryx, Mountain View, CA). After stimulation, medium was centrifuged (800g 4°C, 7 min) and stored at -80°C.

Kumar J.D., Holmberg C., Balabanova S., Borysova L., Burdyga T., Beynon R., Dockray G.J, & Varro A. (2015). Mesenchymal Stem Cells Exhibit Regulated Exocytosis in Response to Chemerin and IGF. PLoS ONE, 10(10), e0141331.

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Multimodal Mitochondrial Labeling

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MitoTracker Red CMXRos and Tf-Alexa Fluor 594 was from Thermo Fisher Scientific. PAO and nocodazole were from Merck. Rapamycin, cycloheximide, and R59949 were from Sigma-Aldrich. Brefeldin A was from Epicenter Technologies.

Mahajan D., Tie H.C., Chen B, & Lu L. (2019). Dopey1-Mon2 complex binds to dual-lipids and recruits kinesin-1 for membrane trafficking. Nature Communications, 10, 3218.

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Immunofluorescence Assay with Inhibitors

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Tunicamycin and TCA were from Sigma-Aldrich (St. Louis, MO), brefeldin A was from Epicentre (Madison, WI), and ionomycin and rapamycin were from Calbiochem (San Diego, CA). PMA was from Sigma-Aldrich, and EGF receptor inhibitor AG1478 was from Cell Signaling (Danvers, MA). Polyclonal antibodies against GFP and cyclophilin B were from Invitrogen (Carlsbad, CA) and Abcam (Cambridge, MA), respectively. The polyclonal fibronectin antibody was from Millipore (Billerica, MA). Alexa Fluor 568–coupled secondary antibody was purchased from Life Technologies (Invitrogen).

Guzmán-Hernández M.L., Potter G., Egervári K., Kiss J.Z, & Balla T. (2014). Secretion of VEGF-165 has unique characteristics, including shedding from the plasma membrane. Molecular Biology of the Cell, 25(7), 1061-1072.

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Quantifying IFN-γ in IAV Peptide-Stimulated CD8+ T Cells

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All IAV peptides (D^bNP_366–374, D^bPA_224–233, K^bPB1_703–711, and D^bPB1‐F2_62–70) were synthesized at the Hartwell Center, St. Jude Children's Research Hospital. Lymphocytes were cultured for 5 h in a 96 well round bottom plate at a concentration of 5–10 × 10⁵ cells per well in 200 μL of RP10 containing 5 mg mL⁻¹ brefeldin A (Epicenter) and 1 μm of peptide, or without peptide for unstimulated controls. After culture, cells were stained with mAb specific for CD8α (53–6.7), CD25 (PC61), CD43(1B11), and mouse CD16/CD32 then fixed in 1% formaldehyde in PBS for 30 min at room temperature. Following fixation, the cells were resuspended in 0.5% saponin (Sigma) for 10 min, incubated with fluorescently labeled anti‐IFN‐γ (XMG1.2) for 30 min at 4°C, followed by flow cytometric analysis.

Keating R., Morris M.Y., Yue W., Reynolds C.E., Harris T.L., Brown S.A., Doherty P.C., Thomas P.G, & McGargill M.A. (2018). Potential killers exposed: tracking endogenous influenza‐specific CD8+ T cells. Immunology and Cell Biology, 96(10), 1104-1119.

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Immunofluorescence and Western Blotting Protocols

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Mouse anti-Lamp1 mAb (H4A3; 1:500 for immunofluorescence or hereafter IF), mouse anti-CD63 mAb (H5C6; 1:100 for IF), and mouse anti-CD8a mAb (OKT8; 1:500 for IF) were from the Developmental Studies Hybridoma Bank. Mouse anti-GFP mAb (catalog no. sc-9996; 1:1000 for Western blotting) was purchased from Santa Cruz Biotechnology, Inc. Mouse anti-EEA1 mAb (catalog no. 610456; 1:500 for IF) and mouse anti-GM130 mAb (catalog no. 610823; 1:500 for IF) were from BD Biosciences. Horseradish peroxidase–conjugated goat anti-mouse (catalog no. 176516; 1:10,000 for Western blotting) was from Bio-Rad. Alexa Fluor–conjugated goat antibodies against mouse IgG (1:500 for IF) were from Thermo Fisher Scientific.
PNGase F was purchased from New England Biolabs. The following small molecules were commercially available: nocodazole (Merck; working concentration: 33 μm), brefeldin A (Epicenter Technologies; working concentration: 10 µg/ml), cycloheximide (Sigma–Aldrich; working concentration: 10 µg/ml), and GalNAc-O-Bn (Sigma–Aldrich; working concentration: 2 mm).

Sun X., Tie H.C., Chen B, & Lu L. (2020). Glycans function as a Golgi export signal to promote the constitutive exocytic trafficking. The Journal of Biological Chemistry, 295(43), 14750-14762.

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Antibody Sources for Cellular Protein Analysis

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Antibodies against the following proteins were from the following sources: actin and Myc (Santa Cruz Biotechnology); HA (Roche); GM130 and CD8 (BD); mouse TMEM115 (Abnova); TMEM115, TGN46, FLAG and FLAG-conjugated agarose beads (Sigma-Aldrich); β-COP (Thermo Scientific); Golgin97 and COG3 were in-house antibodies (Loh and Hong, 2004 (link); Lu et al., 2004 (link)). Fluorochrome-conjugated secondary antibodies were from Invitrogen and horseradish peroxidase (HRP)-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc. Brefeldin A was from Epicentre Biotechnologies. Digitonin and agarose-bound lectins were purchased from Sigma-Aldrich; fluorescent-dye-conjugated lectins were from Vector Laboratories. The TMEM115 human cDNA ORF (accession number NM_007024) was obtained from OriGene USA. GFP–ERGIC53 plasmid was a kind gift from Hans-Peter Hauri (University of Basel, Switzerland).

Ong Y.S., Tran T.H., Gounko N.V, & Hong W. (2014). TMEM115 is an integral membrane protein of the Golgi complex involved in retrograde transport. Journal of Cell Science, 127(13), 2825-2839.

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Flow Cytometric Analysis of Antigen-Specific CD4+ T Cells

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DO11.10 T cells were detected by staining with KJ1–26 TCR clonotypic Ab (eBioscience), and anti-CD4 mAb (clones GK1.5 or RM4–5, BD or eBioscience), after blocking Fc receptors with anti-CD16/CD32. Directly conjugated mAbs were used to detect CD62L (clone MEL-14), CD44 (clone IM7), CD25 (clone PC61.5), GATA-3 (TWAJ) and Thy1.1 (clone HIS51) (all from eBioscience or BD). IL-7Rα was detected by staining with anti-CD127-biotin (clone A7R34, eBioscience) and streptavidin-PE or -APC (eBioscience). Fluorescence intensities of stained cells were measured with a FACScalibur or LSRII flow cytometer and data analyzed with CellQuest or FlowJo software. For intracellular cytokine staining, splenocytes were restimulated ex vivo for 4 h with 1 μg/ml OVA peptide in the presence of 10 μg/ml BrefeldinA (Epicentre Biotechnologies) for the last 3 h and stained with PE-conjugated anti-IL-2 (clone JES6-SH4), -IL-4 (clone 11B11) and -IFN-γ (clone XMG1.2) mAbs (eBioscience or BD Pharmingen), using a Cytofix/Cytoperm kit (BD) according to the manufacturer’s instructions. To follow cell division during priming, naïve CD4⁺ T cells were labeled with 1 μM CFSE (Molecular Probes) for 10 min at room temperature. On day 4 after adoptive transfer and priming, CFSE content of KJ1–26⁺CD4⁺ cells was determined by flow cytometry.

Fleury M., Vazquez-Mateo C., Hernandez-Escalante J, & Dooms H. (2021). PARTIAL STAT5 SIGNALING IS SUFFICIENT FOR CD4+ T CELL PRIMING BUT NOT MEMORY FORMATION. Cytokine, 150, 155770.

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Visualizing Lysosomal and Endosomal Markers

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Mouse anti-Lamp1 monoclonal antibody (mAb) (H4A3; 1:500 for immunofluorescence or hereafter IF), mouse anti-CD63 mAb (H5C6; 1:100 for IF), and mouse anti-CD8a mAb (OKT8; 1:500 for IF) were from Developmental Studies Hybridoma Bank. Mouse anti-GFP mAb (#sc-9996; 1:1000 for Western blot) was purchased from Santa Cruz. Mouse anti-EEA1 mAb (#610456; 1:500 for IF) and mouse anti-GM130 mAb (#610823; 1:500 for IF) were from BD Biosciences. Horseradish peroxidase (HRP)conjugated goat anti-mouse (#176516; 1:10,000 for Western blot) was from Bio-Rad. Alexa Fluorconjugated goat antibodies against mouse IgG (1:500 for IF) were from Thermo Fisher Scientific.
PNGase F was purchased from New England Biolab. The following small molecules were commercially available: nocodazole (Merck; working concentration: 33 µM), brefeldin A (Epicenter Technologies; working concentration: 10 µg/ml), cycloheximide (Sigma Aldrich; working concentration: 10 µg/ml), GalNAc-O-Bn (Sigma Aldrich; working concentration: 2 mM).

Sun X., Tie H.C., Chen B., & Lü L. (2020). Glycans function as a Golgi export signal to promote the constitutive exocytic trafficking.

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