For this purpose, we decided to quantify the intracellular expression of three of the most important proinflammatory cytokines (IL-1β, IL-6, and TNF-α), after TLR stimulation. Cells collected in sodium heparin tubes were polyclonally stimulated for 18 h, with different agonists for human TLR1–9 (InvivoGen, San Diego, CA, USA), in the presence or absence of Brefeldine A (Sigma-Aldrich, St. Louis, MO, USA) in polypropylene tubes. As a negative control, cells were incubated in identical medium without stimulus. After culture, cells were permeabilized with FACS permeabilizing solution (BD Biosciences) and intracellularly stained with PE-labeled mAb against cytokines (IL-1β, IL-6, TNF-α; BD Biosciences) and analyzed by flow cytometry. Percentage of intracellular cytokine-producing B cells, T cells, and monocytes was analyzed using the CellQuest Pro software (BD Biosciences), as previously described [44 (link)].
Brefeldine a
Manufactured by Merck Group
Sourced in United States
Brefeldine A is a chemical compound used in laboratory research. It functions as a tool for studying cellular processes.
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Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (2 mentions)
Facs permeabilizing solution, by BD (1 mentions)
Cellquest pro software, by BD (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Facs lysing solution, by BD (1 mentions)
Anti cd69 percp clone l78, by BD (1 mentions)
Anti cd8 apc h7 clone sk1, by BD (1 mentions)
Anti cd45ra pe cy7, by BD (1 mentions)
Saponin, by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
Q vd oph, by MP Biomedicals (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Mg132, by Merck Group (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Thapsigargin, by Merck Group (1 mentions)
Tunicamycin, by Merck Group (1 mentions)
Anti cd3 cd28, by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
9 protocols using brefeldine a
1
Quantifying Cytokine Profiles in Immune Cells
2
Macrophage Cytokine Modulation by GPR55
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Control THP-1 and foam cells, primed overnight with LPS and untreated or incubated with GPR55 agonist or antagonist (alone or in combination), were washed 3 times with PBS, collected by means of accutase and then stained with FITC-conjugated anti-CD36 specific antibody. Intracellular staining for tumor necrosis factor α (TNF-α) and IL-10 was performed prior to cytokine inhibition, by adding 1 μg/ml brefeldine A (Sigma) 4h before cell collection. Cells were then fixed with 4% formaldehyde (Sigma) for 10 min at room temperature, and were stained in saponine 0.5% with anti-TNF-α APC and anti-IL10 PE. Flow cytometry as also used to assess GPR55 expression by staining macrophages and foam cells with GPR55 antibody both at cell surface or intracellulary upon cell permeabilization with saponine 4% formaldehyde and 0.5% saponine. Flow cytometry analysis was performed on a FACSCanto Flow Cytometer (Beckton Dickinson, NJ, USA), as reported [31 (link)].
3
Quantifying NAGLU-specific Effector T Cells
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In order to determine the frequency of NAGLU-specific effector T cells, 700 µl of whole blood were stimulated for 6 hours at 37°C with NAGLU at 0.5 µg/ml, 1µg/ml or 2 µg/ml, or NAGLU peptide at 0.5 µg/ml or 1µg/ml in the presence of 1µg/ml CD28/CD49d mAbs and 10µg/ml Brefeldine A (Sigma-Aldrich, Lyon France). SEB (1 µg/ml) was used as a positive control. Blood samples were then treated with 10 fold diluted FACS Lysing solution (Becton Dickinson) and stored at -80°C until the ICS assay. Staining was performed on unfrozen blood samples, washed with PBA and permeabilized with BD FACS™ Permeabilizing Solution 2 (Becton Dickinson). The following mAbs were used: anti-CD3-V500 (clone UCHT1, BD Horizon), anti-CD4-APC (clone RPA-T4, BD Pharmingen), anti-CD8-APC-H7 (clone SK1, BD Pharmingen), anti-IFNγ-AF488 (clone B27, BD Pharmingen), anti-CCR7-PE (clone 3D12, BD Pharmingen), anti-CD45RA-PE-Cy7(BD Pharmingen), anti-IL2-PE-CF594 (clone 5344.111, BD Horizon), anti-CD69-PerCP (clone L78, BD Biosciences), and anti-TNFα-V450 (BD Horizon). Staining was done in PBA containing 0.05% saponin (Sigma-Aldrich, Lyon France), cells were fixed in 1% PFA solution and immediately acquired on CyAn Beckman Coulter. The frequency of T cells expressing CD69 and intracellular cytokines was determined with FlowJo software.
4
Th1 Polarization of Naïve CD4 T Cells by HIV-1-Infected iDCs
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Example 13
Naïve CD4 T cells (106/ml) were cocultured for 8 days in the presence of uninfected or HIV-1-infected iDC (106/ml) and resting or activated NK cells (2×105/ml) and tested for Th1 polarization by flow cytometry, as previously reported 56. Briefly, brefeldine A (10 μg/ml) (Sigma Aldrich) was added during the last 16 h of the culture to inhibit protein secretion. Surface staining was performed with PerCP-conjugated CD8 antibodies and FITC-conjugated CD3 antibodies (BD Biosciences, San Jose, Calif.), followed by cell fixation for 15 minutes at 4° C. with 1% PFA and permeabilization with saponin buffer (PBS-BSA 0.2%-NaN3 0.01%-saponin 0.5%), and intracellular staining was performed with APC-conjugated IFNγ- or IL-4-specific antibodies (BD Biosciences, San Jose, Calif.). Stained cells were immediately acquired on a FACScalibur (Becton Dickinson) and analyzed with Flow Jo software. In order to analyze the influence of HIV-1 replication on Th1 polarization, AZT 1 mM was added at the initiation of the culture of naïve CD4 T cells incubated alone, or in the presence of HIV-1 infected iDCs+/−rNK or aNK cells. AZT was left until the end of the coculture. HIV-1-infection of iDCs was performed as described above, in the absence of AZT.
5
Leukemia and Lymphoma Cell Culture
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2-deoxy-d -glucose (2DG), mannose (Mn), thapsigargin (Thps), tunicamycin (Tun), brefeldine A (BFA), cycloheximide (CHX), and MG132 were from Sigma-Aldrich (St. Louis, MO, USA). Q-VD-OPh was from MP Biomedicals Australasia. Cell lines from human leukemias (NALM-6, HL-60, REH, RS4;11, and K562) or lymphomas (JURKAT, SUDHL-4, and RAJI) were cultured at 37 °C under 5% CO2 atmosphere in RPMI-1640 (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS) from Sigma-Aldrich and 2 mM GlutaMAX (Thermo Fisher Scientific). Unless otherwise indicated, cultures were maintained by seeding at 4 × 105 to 1 × 106 cells/mL twice a week.
6
Cytokine Release Assay for PBMCs
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PBMCs were TCR-stimulated for 16 h with soluble 2 μg/mL anti-CD3 and 2 μg/mL anti-CD28 (both Sanquin), 10 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 μg/mL ionomycin (both Sigma-Aldrich), or left untreated. To measure intracellular cytokines, 10 μg/mL brefeldine A (Sigma-Aldrich) was added 1 h after addition of anti-CD3/CD28 or PMA/ionomycin. After incubation and cellular staining, cells were acquired on a Fortessa Cell Analyzer.
7
Characterization of Infiltrating T-cell Subsets
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Transplanted aortas were harvested 3 weeks posttransplantation. Leukocytes were retrieved from the aorta as previously described.18 Briefly, harvested aortas were digested in a phosphate‐buffered saline solution with 450 U/mL collagenase type I, 125 U/mL collagenase type XI, 60 U/mL hyaluronidase type I‐S, and 60 U/mL deoxyribonuclease I for an hour at 37°C. After passing through a 70 µm cell strainer, cell suspension was stimulated O/N with Phorbol 12‐myristate 13‐acetate (Sigma, St. Louis, MO) 10 ng/mL and Ionomycine (EMD Millipore, Burlington, MA) 1 µg/mL O/N at 37°C followed by Brefeldine A (Sigma) 5 µg/mL treatment for 3 hours at 37°C. Infiltrating T cells subsets were characterized using CD3 APC/Fire750 (Biolegend, San Diego, CA), anti‐TCR gamma delta (TCRγδ) (clone GL3), BV421 (BD Biosciences), TCR beta (TCRβ) chain (clone: H57‐597), Alexa Fluor 488 (BD Biosciences), transcription factor retinoic acid receptor‐related orphan receptor gamma (RORγ) PerCP‐efluor 710 (eBioscience, San Diego, CA), IL‐17A BV711 (Biolegend), and IL‐17F Alexa fluor 647 (Biolegend) antibody staining. Samples were run on a flow cytometer (FACScan, Becton Dickinson) and analyzed on the computer software FlowJo.
8
Cellular Activation and Proliferation
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Cell media and CFSE were purchased from Life Technologies (Carlsbad, CA). Purified antibodies or fluorochrome labeled antibodies were purchased from eBioscience (San Diego, CA). Brefeldine A, Giemsa stain and CQ were purchased from Sigma (St. Louis, MO).
9
Leukocyte Cytokine Response to TLR7 Agonist
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Collected blood samples were distributed in 1.5 mL polypropylene tubes and supplemented with 200 μL of RPMI medium containing 10% of fetal bovine serum (FBS). Samples were cultured in the presence of Resiquimod (R848, 1 μg/mL, Invivogen) to simulate TLR7 signaling pathway. Samples cultured in absence of stimulus were used as controls. After 1h of incubation at 37°C 5% CO2, Brefeldine A (10 μg/mL, Sigma) was added to repress cytokine release. Four hours later, samples were incubated for 10 minutes with ammonium chloride in order to perform the lysis of red cells. Staining was performed after one wash with DPBS 1x (Gibco) on whole leukocytes.
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