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Brefeldin a

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Brefeldin A is a chemical compound that functions as an inhibitor of protein transport from the endoplasmic reticulum to the Golgi apparatus in eukaryotic cells. It is commonly used as a research tool in cell biology to study intracellular protein trafficking.

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Lab products found in correlation

Monensin, by BD (120 mentions) Ionomycin, by Merck Group (118 mentions) Cytofix cytoperm kit, by BD (68 mentions) Phorbol myristate acetate (pma), by Merck Group (61 mentions) Cytofix cytoperm, by BD (37 mentions) Lsrii flow cytometer, by BD (23 mentions) Facscanto 2, by BD (22 mentions) Lsrfortessa, by BD (22 mentions) Golgistop, by BD (21 mentions) Ionomycin, by BD (19 mentions) Phorbol 12 myristate 13 acetate, by Merck Group (19 mentions) Lsrfortessa flow cytometer, by BD (18 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (17 mentions) L glutamine, by Thermo Fisher Scientific (14 mentions) Facscalibur, by BD (13 mentions) Perm wash buffer, by BD (12 mentions) Facscanto 2 flow cytometer, by BD (12 mentions) Cytofix cytoperm solution, by BD (11 mentions) Anti ifn γ apc, by BD (11 mentions) Phorbol 12 myristate 13 acetate pma, by Merck Group (11 mentions)

Close spelling variants of this product

Brefeldin A solution (8 citations) GolgiPlug containing brefeldin A (25 citations) Brefeldin A (GolgiPlug, (57 citations) Protein Transport Inhibitor (Containing Brefeldin A) (3 citations) BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit with BD GolgiPlug Protein transport inhibitor containing Brefeldin A (2 citations) Fast Immune Brefeldin A Solution (3 citations) Peptide pool and brefeldin A (2 citations) BD GolgiPlug with brefeldin A (2 citations) Containing Brefeldin A (3 citations) Brefeldin A (BFA, (10 citations)

612 protocols using brefeldin a

1

HIV Gag Peptide Stimulation Assay

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PBMCs were incubated for 6 hours with an HIV Gag peptide pool at a concentration of 0.5μg/ml/peptide in the presence of 2.5 μg/ml brefeldin A (BD GolgiPlug) and (0.3μl/ml) monensin (BD GolgiStop). For delayed ICS assays, 2.5 μg/ml brefeldin A (BD GolgiPlug) and (0.3μl/ml) monensin (BD GolgiStop) were added 9h after stimulation with the Gag peptide pool and cells were cultured for 12h. Unstimulated cells were used as negative control and SEB (0.5) μg/ml as positive control. Cells were then stained with viability dye (20 min at 4 C°), surface markers (20 min at 4 C°), fixed with Fixation Solution (eBioscience), and stained for intracellular proteins in permeabilization 1X buffer (eBioscience) (30 min at 4 C°) as described in Supplementary Tables 15 and 16. Cells were acquired on an LSR II (BD Biosciences).
Morou A., Brunet-Ratnasingham E., Dubé M., Charlebois R., Mercier E., Darko S., Brassard N., Nganou-Makamdop K., Arumugam S., Gendron-Lepage G., Yang L., Niessl J., Baxter A.E., Billingsley J.M., Rajakumar P.A., Lefebvre F., Johnson R.P., Tremblay C., Routy J.P., Wyatt R.T., Finzi A., Douek D.C, & Kaufmann D.E. (2019). Altered differentiation is central to HIV-specific CD4+ T cell dysfunction in progressive disease. Nature immunology, 20(8), 1059-1070.
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2

Cytokine Profiling of Sorted Immune Cells

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Antibody clones are listed in the Supplemental Experimental Procedures. For staining of ROS, CellROX Deep Red reagent (Life Technologies) was used. For intracellular cytokine staining, T cells were incubated 4 hr at 37 C in RPMI containing 10% FCS with PMA (50 ng/mL), ionomycin (500 ng/mL), and Brefeldin A (BD); myeloid cells with Brefeldin A (BD). Flow cytometric analysis was carried out using a FACSCanto II (BD) or a LSR II Fortessa (special order research product, BD) with FACS Diva Software.
Cell sorting is described in detail in the Supplemental Experimental Procedures.
RNA Isolation and Quantitative RT-PCR Total RNA was isolated from sorted microglia using the RNeasy Plus Micro kit (QIAGEN) and cDNA was generated using SuperScript II (Invitrogen) according to manufacturer's instructions. Gene expression was measured by qPCR analysis using the CFX 384 system (Bio-Rad) with SYBR Green Supermix (Bio-Rad). Primer sequences can be found in the Supplemental Experimental Procedures. Transcript expression was normalized to Polr2a.
Spath S., Komuczki J., Hermann M., Pelczar P., Mair F., Schreiner B, & Becher B. (2017). Dysregulation of the Cytokine GM-CSF Induces Spontaneous Phagocyte Invasion and Immunopathology in the Central Nervous System. Immunity, 46(2).
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3

Quantification of Virus-Specific CD8+ T Cells

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Thy1.1/1.1 or Thy1.1/1.2 P14 CD8+ T cells were obtained from spleens or peripheral blood of young naïve P14 mice and 103 cells were injected i.v. into naïve C57BL/6 (Thy1.2/1.2) recipients (45 (link)). Lymphocytes were isolated from the spleen or peripheral blood as indicated and LCMV-specific CD8+ T cells were identified by tetramer staining for endogenous GP276- and GP33-specific CD8+ T cells or Thy1.1-staining for P14 TCR-transgenic cells in inbred C57BL/6 mice, or CD11a CD8α-staining for CD8+ T cells in outbred NIH Swiss mice (53 (link)). Inhibitory molecule expression (PD-1, LAG-3, and 2B4) was determined on virus-specific CD8+ T cells. The frequency of CD8+ T cells producing cytokine after stimulation with indicated peptides was determined using intracellular cytokine staining (IFNγ, TNFα) after 5h incubation at 37°C in the presence of Brefeldin A (BD Biosciences) (54 (link)). Cytokine production (IFNγ) from the peripheral blood, was determined after 5h stimulation at 37°C with indicated peptides in the presence of Brefeldin A (BD Biosciences) and EL4 (105) antigen presenting cells as previously described (47 (link)). FlowJo software (Tree Star) was used for analysis of samples acquired on a Canto flow cytometer (BD Biosciences).
Condotta S.A., Khan S.H., Rai D., Griffith T.S, & Badovinac V.P. (2015). Poly-microbial sepsis increases susceptibility to chronic viral infection and exacerbates CD8+ T cell exhaustion. Journal of immunology (Baltimore, Md. : 1950), 195(1), 116-125.
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4

Cytokine Profile in Viral Hepatitis

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PBMCs from HD, HCV without cryoglobulinemia, and HCV-CV patients were stimulated for 4 hours with 50 ng/mL phorbol myristate acetate and 1 mmol/L ionomycin (Sigma-Aldrich, Saint-Louis, MO) in the presence of brefeldin A (BD PharMingen, Le Pont de Claix, France). Cells cultured in the presence of brefeldin A were stained for cell surface markers and then permeabilized with Cytofix/Cytoperm buffer (BD PharMingen) and stained with fluorescein isothiocyanateconjugated interferon (IFN)g (BD PharMingen), Alexa Fluor 647-conjugated interleukin (IL)17A (eBioscience), and Alexa Fluor 647-conjugated IL21 (BioLegend). Th1 were identified as IFNgþCD4þ cells in CD3þ T lymphocytes. Th17 were identified as IL17AþCD4þ cells in CD3þ T lymphocytes. Follicular T-helper (Tfh) cells were identified as CD4þCXCR5þIL21þ cells in CD3þ T lymphocytes.
The levels of the following serum cytokines were measured using Human Cytokine MILLIPLEX according to the recommendations of the manufacturer (Merck Millipore, Billerica, MA): IFNg, tumor necrosis factor (TNF)a, IL12p70, IL4, IL5, IL10, IL13, IL17A, and IL21. The levels of BAFF (R&D Systems, Abingdon, United Kingdom) were determined by enzymelinked immunosorbent assay.
Comarmond C., Garrido M., Pol S., Desbois A.C., Costopoulos M., Le Garff-Tavernier M., Si Ahmed S.N., Alric L., Fontaine H., Bellier B., Maciejewski A., Rosenzwajg M., Klatzmann D., Musset L., Poynard T., Cacoub P, & Saadoun D. (2017). Direct-Acting Antiviral Therapy Restores Immune Tolerance to Patients With Hepatitis C Virus-Induced Cryoglobulinemia Vasculitis. Gastroenterology, 152(8).
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5

HIV Gag Peptide Stimulation Assay

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PBMCs were incubated for 6 hours with an HIV Gag peptide pool at a concentration of 0.5μg/ml/peptide in the presence of 2.5 μg/ml brefeldin A (BD GolgiPlug) and (0.3μl/ml) monensin (BD GolgiStop). For delayed ICS assays, 2.5 μg/ml brefeldin A (BD GolgiPlug) and (0.3μl/ml) monensin (BD GolgiStop) were added 9h after stimulation with the Gag peptide pool and cells were cultured for 12h. Unstimulated cells were used as negative control and SEB (0.5) μg/ml as positive control. Cells were then stained with viability dye (20 min at 4 C°), surface markers (20 min at 4 C°), fixed with Fixation Solution (eBioscience), and stained for intracellular proteins in permeabilization 1X buffer (eBioscience) (30 min at 4 C°) as described in Supplementary Tables 15 and 16. Cells were acquired on an LSR II (BD Biosciences).
Morou A., Brunet-Ratnasingham E., Dubé M., Charlebois R., Mercier E., Darko S., Brassard N., Nganou-Makamdop K., Arumugam S., Gendron-Lepage G., Yang L., Niessl J., Baxter A.E., Billingsley J.M., Rajakumar P.A., Lefebvre F., Johnson R.P., Tremblay C., Routy J.P., Wyatt R.T., Finzi A., Douek D.C, & Kaufmann D.E. (2019). Altered differentiation is central to HIV-specific CD4+ T cell dysfunction in progressive disease. Nature immunology, 20(8), 1059-1070.
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6

Quantifying Cytokine Responses to EBV Antigens

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CD8-depleted PBMCs from DR7+ healthy carriers were stimulated with the EBNA2276–295 peptide at a concentration 0.005 μg/ml for 4 h in the presence of brefeldin A (BD Biosciences). EBV-transformed autologous B lymphoblastoid cell lines (LCLs) were generated as described previously (30 (link)). Bulk PBMCs from healthy carriers or patients with IM were stimulated with autologous LCLs at a ratio of 1:1 for 16 h in the presence of brefeldin A, monensin, and anti–CD107a-FITC (H4A3) (BD Biosciences). Stimulated cells were washed twice in R8 and processed as described above prior to flow cytometric analysis of CD107a, IFN-γ, IL-2, and TNF-α.
Meckiff B.J., Ladell K., McLaren J.E., Ryan G.B., Leese A.M., James E.A., Price D.A, & Long H.M. (2019). Primary EBV Infection Induces an Acute Wave of Activated Antigen-Specific Cytotoxic CD4+ T Cells. The Journal of Immunology Author Choice, 203(5), 1276-1287.
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7

Quantification of Antigen-Specific T Cells

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For the detection of OVA-, TRP-2, gp100-specific IFNγ producing CD8+ T cells, lymphocytes were re-stimulated with 1.0 μg/mL OVA257-264, TRP-2180-188, gp10025-32, respectively in the presence of 5 μg/ml Brefeldin A (BD PharMingen) for 4–5 h. To address the presence of polyclonal IFNγ producing CD8+ and CD4+ T cells, cells were restimulated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA), 10 μg/ml ionomycin and 5 μg/ml Brefeldin A. IFNγ production by OVA-specific CD4+ T cells was analyzed upon two day re-stimulation with OVA262-276.
Unger W.W., Mayer C.T., Engels S., Hesse C., Perdicchio M., Puttur F., Streng-Ouwehand I., Litjens M., Kalay H., Berod L., Sparwasser T, & van Kooyk Y. (2014). Antigen targeting to dendritic cells combined with transient regulatory T cell inhibition results in long-term tumor regression. Oncoimmunology, 4(8), e970462.
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8

Cytokine Profiling of MHV-Specific T Cells

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Splenocytes were restimulated ex vivo in complete media with either 30nM p79 MHV-specific peptide or with 1 μg/ml PMA and 1 μg/ml Ionomycin (Sigma Life Sciences) in the presence of Brefeldin A (BD Biosciences) at 37°C for 4h. Subsequently, cells were surface stained followed by fixation with the Fix/Perm intracellular staining kit (BD Pharmingen) at 4°C for 20 min, and then stained with antibodies against IFN-γ (PE-Dazzle, BioLegend, clone XMG1.2), and TNF (APC, Biolegend, clone MP6-XT22) per manufacturer’s instructions. For assessment of degranulation activity, an anti-CD107a antibody (BV421, Biolegend, clone 1D4B) was added along with the stimuli outlined above, in the presence of Brefeldin A and Monensin (BD Biosciences) and cultured at 37°C for 4h followed by surface staining at 4°C for 20 min.
Crepeau R.L., Elengickal J.A., La Muraglia GM 2.n.d, & Ford M.L. (2019). Impact of Selective CD28 Blockade on Virus-Specific Immunity to a Murine EBV Homolog. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 19(8), 2199-2209.
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9

NK Cell Stimulation and Functional Assays

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CD49b+ NK cells were enriched by magnetic sorting (Miltenyi) from BM. For stimulation through NK1.1, 10 µg/ml anti-NK1.1 (PK136, 108701; BioLegend) in NaHCO3 (pH 9.2) was precoated on an ELISA plate (655081; Greiner Bio-One). After culture in 20 ng/ml murine IL-15 (210-15; PeproTech) overnight, NK cells were then added to the plate with Brefeldin A (555029; BD Biosciences) for the last 4.5 h. For stimulation through cytokines, NK cells were cultured with 1,000 U/ml IL-2 (212-12; PeproTech) and 10 ng/ml IL-12 (210-12, PeproTech) overnight and Brefeldin A for the last 4.5 h. Cytofix/Cytoperm Fixation/Permeabilization Kit (554714; BD Biosciences) was used to detect IFN-γ (XMG1.2, 12-7311-81; eBioscience), and Cyto-Fast Fix/Perm Buffer Set (426803; BioLegend) was used to detect GZMB (QA16A02, 372207; BioLegend) according to the manufacturer’s instructions.
Li Z.Y., Morman R.E., Hegermiller E., Sun M., Bartom E.T., Maienschein-Cline M., Sigvardsson M, & Kee B.L. (2021). The transcriptional repressor ID2 supports natural killer cell maturation by controlling TCF1 amplitude. The Journal of Experimental Medicine, 218(6), e20202032.
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10

Intracellular Cytokine Profiling of Lung and Bone Marrow Immune Cells

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To measure IL‐5 and IL‐13, 5 × 106 lung cells were re‐stimulated for 4 hours (at 37° C 5% CO2) with either 1% BSA RPMI media alone containing Brefeldin/A (BD Biosciences); 1% BSA RPMI media containing 50 ng/ml PMA and 500 ng/ml Ionomycin from Sigma‐Aldrich (St. Louis, Missouri) and Brefeldin/A or 40 ng/ml IL‐33 from R&D systems (Minneapolis, Minnesota) and Brefeldin/A. Intracellular cytokine staining was done using the Cytofix/Cytoperm kit from BD Biosciences.
To stimulate bone marrow ILC2s with cytokines, 5–10 million bone marrow cells were stimulated for 13–16 hours (at 37° C 5% CO2) with 1% BSA RPMI, 50μM β‐mercaptoethanol and 1% pen/strep containing either 10 ng/ml recombinant murine IL‐7 from Peprotech (Cranbury, NJ) or IL‐2 (Peprotech) in combination with anti‐IL‐2 mAb [S4B6] (ThermoFisher, catalog no. 16‐7020‐85). Intracellular staining was performed using the Fixation and Cell Permeabilization kit (ThermoFisher).
Sheikh A., Lu J., Melese E., Seo J.H, & Abraham N. (2022). IL‐7 induces type 2 cytokine response in lung ILC2s and regulates GATA3 and CD25 expression. Journal of Leukocyte Biology, 112(5), 1105-1113.
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