Cytokine production was analyzed in samples from in vitro polarization, OVA restimulation and lungs from airway-inflammation experiments. Single cell suspensions were seeded in a 96-well plate and restimulated with a mix containing 25 ng/ml phorbol-12-myristat-13-acetat (PMA), 500 ng/ml ionomycin and 10 µg/ml brefeldin A (BFA; all Merck) for 5 hours at 37°C, 5% CO2. Afterwards, cells were harvested and surface staining was performed with anti-CD4 and fixable viability stain 780 followed by fixation as described above. Intracellular staining was performed with antibodies directed against IL-4, IL-13 and IFN-γ. Samples were measured on a LSRFortessa flow cytometer (BD Biosciences). Flow cytometry data was analyzed with FlowJo v10 (BD Biosciences).
Brefeldin a bfa
Manufactured by Merck Group
Sourced in United States, Switzerland, United Kingdom
Brefeldin A (BFA) is a lactone compound that is produced by several fungal species. It is a useful tool for cell biology research as it disrupts the Golgi apparatus, leading to the accumulation of secretory proteins in the endoplasmic reticulum.
Automatically generated - may contain errors
Lab products found in correlation
Ionomycin, by Merck Group (35 mentions)
Phorbol myristate acetate (pma), by Merck Group (21 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (5 mentions)
Lsrfortessa, by BD (4 mentions)
Cycloheximide (chx), by Merck Group (4 mentions)
Cytofix cytoperm kit, by BD (4 mentions)
Cytexpert 1, by Beckman Coulter (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
Thapsigargin, by Merck Group (3 mentions)
Tunicamycin tm, by Merck Group (3 mentions)
Thapsigargin tg, by Merck Group (3 mentions)
Facsaria, by BD (3 mentions)
Nocodazole, by Merck Group (3 mentions)
Cellquest software, by BD (3 mentions)
Facscalibur, by BD (3 mentions)
Saponin, by Merck Group (3 mentions)
Beckman cytoflex, by Beckman Coulter (3 mentions)
Fixation and permeabilization solution, by BD (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
135 protocols using brefeldin a bfa
1
Cytokine Production Analysis in Airway Inflammation
2
ONS Cell Culture and Proteomics
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ONS cells were cultured in DMEM/F12 (Thermo Fisher Scientific) supplemented with 10% foetal bovine serum (Thermo Fisher Scientific). For proteomics, cells were grown until confluence in a T75 flask (Nunc) then scraped, pelleted and stored at −80°C until analysed. For qRT‐PCR experiments cells were grown until confluence in six well plates (Nunc) before treatment with Tunicamycin (Sigma), Brefeldin A (BFA, Merck) or Thapsigargin (Sigma).
3
Multiparametric Immune Cell Analysis
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Spleens were collected and a single cell suspension was prepared using 70 µm nylon mesh. Ears were harvested and dorsal and ventral sections were split with forceps. Digestion was performed with 1 mg/ml DNAse I (Sigma) and 1 mg/ml collagenase D (Roche) in HBSS (10 mM Hepes) in a gentleMACS Octo Dissociator (Miltenyi Biotec). Cytokine production was analyzed after restimulation with 25 ng/ml phorbol-12-myristat-13-acetat (PMA), 500 ng/ml ionomycin and 10 µg/ml brefeldin A (BFA) (all Merck) for 4 hours at 37°C, 5% CO2. Surface and intracellular staining was performed as in (26 (link)). Doublet exclusion was performed by gating on SSC-W/SSC-H or SSC-H/SSC-A. The surface staining was performed first along with fixable viability stain 780 (BD Pharmingen) according to the manufacturer’s instructions. The antibodies used are listed in Supplementary Table 2
4
Eosinophil ROS Inhibition and Degranulation
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To block reactive oxygen species (ROS) production, eosinophils were incubated with the NAPDH oxidase inhibitor diphenyleneiodonium chloride (DPI, Calbiochem, Merck Millipore, Darmstadt, Germany) at increasing concentrations of 1-75 µM for 30 min at 37°C. To block degranulation, eosinophils were incubated with 10 µg/ml brefeldin A (BFA, Sigma-Aldrich co, Buchs, Switzerland) which inhibits vesicular transport and granule emptying (24) for 30 min at 37°C. To degrade the DNA scaffold of EETs (5), 100 U/ml of deoxyribonuclease I (DNase I, Worthington Biochemical Corporation, Lakewood, USA) was used. All reagents were added to eosinophils prior to their application on skin sections.
To quantify ROS production, 1 µM dihydro-rhodamine-123 (DHR, Sigma-Aldrich, Buchs, Switzerland) was added to activated eosinophils prior to their addition to human skin sections incubated with BPS, NHS or PBS in a black, glass-bottom 96-well plate (Greiner Bio-One GmbH, Frickenhausen, Germany) (25) . Fluorescence activity of the DHR 123 was measured at excitation 485 nm and the fluorescence emission at 538 nm, using a SpectraMaxM2 plate reader (Bucher Biotech, Basel, Switzerland) over a time period of 2 h.
To quantify ROS production, 1 µM dihydro-rhodamine-123 (DHR, Sigma-Aldrich, Buchs, Switzerland) was added to activated eosinophils prior to their addition to human skin sections incubated with BPS, NHS or PBS in a black, glass-bottom 96-well plate (Greiner Bio-One GmbH, Frickenhausen, Germany) (25) . Fluorescence activity of the DHR 123 was measured at excitation 485 nm and the fluorescence emission at 538 nm, using a SpectraMaxM2 plate reader (Bucher Biotech, Basel, Switzerland) over a time period of 2 h.
5
Inhibitor Treatment of HeLa Cells
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HeLa cells were grown in 24-well plates and treated with different concentrations of the inhibitors TC, 2-FPA and RG (Santa Cruz Biotechnologies) either before or after infection, as indicated in each particular experiment. For TC, 5, 7.5, 10, 15, and 25 μM were used depending on the experiment. For 2-FPA, 25, 50, 100, 150, 200, 250, 300, 350 μM were used depending on the experiment. For RG, 10, 25, 50, 100, 150 μM were used depending on the experiment. For the experiments with Brefeldin A (BFA) (Sigma Aldrich), 10 μM was used and added from 2–4 hpi. Cells were infected with Ct as described previously, and processed either for confocal microscopy or for WB.
6
Human Osteosarcoma Cell Lines Culture
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Human osteosarcoma cell lines (U2OS and MG-63) were purchased from the American Type Culture Collection. The cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO, Los Angeles, CA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), and penicillin–streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37 °C in a humidified atmosphere of 5% CO2. pGL3-Basic, pRL-TK and the Dual luciferase reporter assay system were purchased from Promega (Madison, WI, USA). Brefeldin A (BFA), thapsigargin (TG), and tunicamycin (TM) were obtained from Sigma-Aldrich (St. Louis, MO, USA).
7
Isolation and Activation of T cells
8
Connexin Trafficking and Stabilization
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To block ER-to-Golgi transport, connexin-expressing cells were treated with 5 µg/ml brefeldin A (BFA; Sigma-Aldrich) for up to 18 h in DMEM complete medium at 37°C. To examine the relative half-life of Cx30, REKs were treated with 10 µg/ml of the translational inhibitor cycloheximide (CHX; Sigma-Aldrich). To elucidate the role of cytoskeletal elements in the stabilization of connexin plaques, Cx30-and Cx43-expressing REKs were treated with the microtubule-disrupting drug nocodazole (10 μM; Sigma-Aldrich) or with the actin depolymerization drug cytochalasin B (2.5 µg/ml; Sigma-Aldrich) for up to 10 h in DMEM complete medium at 37°C.
9
FLT3 Inhibitor Screening Protocol
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The polyclonal rabbit anti-FLT3 (C-20) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-FLT3 (Tyr591), anti-phospho-STAT5 (Tyr694), anti-phospho-AKT (Ser473), anti-phospho-p44/42 MAPK (Thr202/Tyr204), anti-phospho-BAD (Ser112), anti-AKT, anti-MAPK, anti-BAD, anti-PIM1 and Sepharose Conjugated Myc-tag antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-STAT5 antibody was from BD Transduction Laboratories (San Jose, CA). Anti-FLAG antibody (clone M2) and anti-FLAG M2 affinity gel were from Sigma-Aldrich (St.Louis, MO). Anti-mouse and anti-rabbit horseradish peroxidase antibodies were purchased from GE Healthcare (Buckinghamshire, UK).
Five FLT3 inhibitors were used in this study: quizartinib was purchased from MedChemExpress (Mommouth Junction, NJ), sorafenib was from Selleck Chemicals (Houston, TX), KW-2449 [33 (link)] was a generous gift from Kyowa Hakko Kirin Co., Ltd. (Tokyo, Japan), midostaurin (PKC412) was from Enzo Life Sciences (Plymouth Meeting, PA), lestaurtinib (CEP701) and U0126 were from Merck Millipore Corporation (Billerica, MA). Tunicamycin and brefeldin A (BFA) were purchased from Sigma-Aldrich. Recombinant human FLT3 ligand (FL) was purchased from R&D Systems, Inc.
Five FLT3 inhibitors were used in this study: quizartinib was purchased from MedChemExpress (Mommouth Junction, NJ), sorafenib was from Selleck Chemicals (Houston, TX), KW-2449 [33 (link)] was a generous gift from Kyowa Hakko Kirin Co., Ltd. (Tokyo, Japan), midostaurin (PKC412) was from Enzo Life Sciences (Plymouth Meeting, PA), lestaurtinib (CEP701) and U0126 were from Merck Millipore Corporation (Billerica, MA). Tunicamycin and brefeldin A (BFA) were purchased from Sigma-Aldrich. Recombinant human FLT3 ligand (FL) was purchased from R&D Systems, Inc.
10
PBMC Activation and Cytokine Analysis
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Peripheral blood mononuclear cells (PBMCs) were isolated from heparin blood by density gradient centrifugation (Ficoll-Hypaque, PLUS Healthcare, Buckinghamshire, UK). PBMCs were washed twice with PBS, resuspended in complete media and stimulated in sterile polystyrene round bottom tubes (Falcon; BD Biosciences). Each tube contained 106 PBMC, anti-CD107a antibody (BD Biosciences), Monensin (‘Golgistop’, BD Biosciences), one of the stimulants (SEB, one of 16 different PepMix solutions, or tuberculin) plus complete media to a final volume of 250 μl. Stimulations were incubated at 37 °C in 5% CO2, humidified atmosphere, for 2 h prior to the addition of 250 μl Brefeldin A (BFA, final concentration 10 μg/ml; Sigma-Aldrich). Incubation was stopped after an additional 14 h.
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