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8 protocols using brefeldin a

1

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.
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2

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.
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3

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).
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4

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

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All IAV peptides (DbNP366–374, DbPA224–233, KbPB1703–711, and DbPB1‐F262–70) 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.
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5

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).
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6

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).
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7

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.
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8

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).
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