Cells were isolated from AMC and Coleman fat at the indicated time points. Briefly, the fat was digested (30 minutes on a shaker at 37° C) in PBS containing 0.075% collagenase. Mature adipocytes and connective tissue were removed by centrifugation at 800 × g for 5 minutes. The cell pellets were resuspended and filtered through a 100 μm mesh and a 70 μm mesh. Total cells were then counted. Then, the cells were stimulated for 4 h with 20 ng/ml phorbol myristate acetate (Sigma-Aldrich) and 1 μg/ml ionomycin (Sigma-Aldrich) prior to addition of 10 μg/ml brefeldin A (BFA) (eBiosciences). For the detection of surface markers, cells were stained with CD31 (eBiosciences), CD34 (eBiosciences), CD90 (eBiosciences), and CD45 (eBiosciences) and then incubated for 15 min at 4° C in the dark. After washing, cells were fixed and permeabilized using fixation buffer and permeabilization buffer (BD Biosciences). Acquisition was performed on a Coulter Epics-XL flow cytometer using the System II software (Coulter Corporation, Brea, CA, USA). Data analysis was performed using FCS express software (De Novo Software, Los Angeles, CA, USA).
Brefeldin a bfa
Manufactured by Thermo Fisher Scientific
Sourced in United States
Brefeldin A (BFA) is a lactone compound used as a research tool in cell biology. It inhibits the Golgi-to-endoplasmic reticulum transport, causing the Golgi apparatus to disassemble and the endoplasmic reticulum to swell. This disruption of intracellular vesicle trafficking can be observed through microscopy techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ionomycin, by Merck Group (8 mentions)
Phorbol myristate acetate (pma), by Merck Group (5 mentions)
Fixable viability dye, by Thermo Fisher Scientific (2 mentions)
Permeabilization buffer, by BD (1 mentions)
Fcs express software, by De Novo Software (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Baf901, by R&D Systems (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Facscan, by BD (1 mentions)
Ionomycin, by Thermo Fisher Scientific (1 mentions)
Cellquest software, by BD (1 mentions)
Fixation buffer, by Thermo Fisher Scientific (1 mentions)
Intracellular staining perm wash buffer, by BioLegend (1 mentions)
Fixation buffer, by BioLegend (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Pma ionomycin, by MultiSciences Biotech (1 mentions)
Il 17, by Thermo Fisher Scientific (1 mentions)
Canto 2 flow cytometer, by BD (1 mentions)
Fetal bovine serum (fbs), by Cytiva (1 mentions)
Opti mem 1 reduced serum media, by Thermo Fisher Scientific (1 mentions)
25 protocols using brefeldin a bfa
1
Isolation and Characterization of Adipose-Derived Cells
2
Secretome Protein Analysis by Western Blot
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Secretomes were probed by Western blotting for selected classically secreted proteins, namely MMP1 (antibody BAF901, R&D Systems, Oxfordshire, UK), MMP3 (antibody AF913, R&D Systems), TGFB/TGFβig-h3 (antibody AF2935, R&D Systems) and SCG2 (antibody ab96589, Abcam, Cambridge, UK). In some experiments, the cells were pre-incubated for 30 min with 10 μg.ml−1 brefeldin A (BFA; eBioscience, Ltd., Hatfield, UK), or 10 μg.ml−1 cycloheximide (Sigma, Dorset, UK) and/or 1 μm ionomycin (Sigma). Proteins were resolved by SDS-PAGE and processed for Western blotting as described previously (13 (link)).
3
Intracellular Cytokine Staining Protocol
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Intracellular cytokine staining was performed as previously described [16 (link)]. In brief, 2.5 × 106 cells from control and NNAV-treated mice were stimulated with Phorbol-12-myristate-13-acetate (PMA, 50 ng/mL final concentration; Sigma, St. Louis, Missouri, USA) plus ionomycin (1 μM; eBioscience, San Diego, CA, USA) and brefeldin A (BFA; 1 : 1000 dilution, eBioscience, San Diego, CA, USA) for 6 h in 24-well plate. After stimulation, cells were collected and stained with anti-mouse CD3e-APC and CD4-PE antibody (eBioscience, San Diego, CA, USA) for 20 min at room temperature. Cells were washed and fixed with fixation buffer (eBioscience, San Diego, CA, USA) for 25 min at room temperature. Fixed cells were washed twice with permeabilization buffer (eBioscience, San Diego, CA, USA) and then intracellular cytokine staining was carried out using FITC conjugated anti-mouse IFN-γ, IL-4, or IL-17 antibody (eBioscience, San Diego, CA, USA) for 20 min. Samples were analyzed with BD FACScan (BD Biosciences, USA) using Cell Quest software (BD Biosciences, USA).
4
Comprehensive Immune Profiling by Flow Cytometry
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First, mononuclear cells were separated from fresh blood samples. Cells were stimulated with PMA/ionomycin (CS1002, Multisciences, China) and Brefeldin A (BFA) (00–4975, eBioscience, USA) and cultured at 37°C in 5% CO2 for 5 h. The cells were then stained with CD45, CD3, CD4, CD8, PD-1, and Tim-3 antibodies for 15 min at room temperature in the dark. The cells were washed twice with Phosphate Buffered Saline (PBS), fixed with Fixation Buffer (420801, BioLegend, USA) in the dark for 15 min, permeabilized with 1X Intracellular Staining Perm Wash Buffer (421002, BioLegend, USA) twice, and then intracellularly stained for IFN-γ in the dark for 15 min. Finally, 30,000 CD3+ cells were acquired for analysis with a BD FACS Canto flow cytometer (BD Biosciences, San Jose, USA) and subsequent analysis using Flowjo software (Flowjo LLC, USA).
Meanwhile, 100 μL of BM or PB blood samples were collected and mixed with Red Blood Cell Lysis Buffer in the dark at room temperature for 8 min. Cell surface staining analysis for CD45, HLA-DR, CD117, CD34, PD-L1, PD-L2 Tim-3, and corresponding isotypes (ISO) and intracellular staining analysis for Gal-9 were performed. The expression was detected and analyzed using flow cytometry and FlowJo software, as mentioned above. The antibodies used in this study are listed in Supplementary Table S2.
Meanwhile, 100 μL of BM or PB blood samples were collected and mixed with Red Blood Cell Lysis Buffer in the dark at room temperature for 8 min. Cell surface staining analysis for CD45, HLA-DR, CD117, CD34, PD-L1, PD-L2 Tim-3, and corresponding isotypes (ISO) and intracellular staining analysis for Gal-9 were performed. The expression was detected and analyzed using flow cytometry and FlowJo software, as mentioned above. The antibodies used in this study are listed in Supplementary Table S2.
5
Cytokine Production Assay Protocol
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IFN‐γ and IL2 production was detected by intracellular staining. Transduced PBMCs, 1.5 × 105 per well, were co‐cultured with target cells, 1 × 105 per well, for 6 h at an E:T (Effector:Target) ratio of 1.5:1. Brefeldin A (“BFA”; eBioscience) was added 2 h into the co‐culture for prevention of cytokine secretion. Cells were then stained for viability with Zombie‐aqua, fixed, permeabilized, and stained for hCD4, hCD8, hIL2, hIFN‐γ, and mTCRβ and analyzed by flow cytometry. Assays were performed in duplicates.
6
Intracellular Cytokine Analysis of Graft-Draining Lymph Nodes
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Cell suspensions were collected from the graft-draining lymph nodes and spleens of graft recipients. Levels of intracellular cytokines were determined after cells were stimulated with 50 ng/ml pyromellitic acid (PMA), 500 ng/ml ionomycin (Sigma-Aldrich) and 3 μg/ml brefeldin A (BFA) (eBioscience, San Diego, CA, USA) for 4 hours in complete medium. Antibodies against mouse CD3, CD4, IFN-γ (BioLegend, San Diego, CA, USA), Foxp3 and IL-17 (eBioscience, San Diego, CA, USA) were used. Cells at 1 × 106 cells/ml were incubated for 20 min with Fc block and then stained for surface markers (CD3 and CD4) for another 25 min on ice in a dark room. IFN-γ, Foxp3 and IL-17 intracellular staining were performed by utilizing eBioscience Fixation/Permeabilization according to the protocols recommended. The stained cells were assessed using a Canto II flow cytometer (BD, Franklin Lakes, NJ, USA) and data were analysed using Flow Jo software (Tree Star, Oregon, USA).
7
Quantifying Extracellular Vesicle Secretion
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Human kidney 293T cells were purchased from Alstem Inc (Richmond, CA). Human liver HepG2 cells were purchased from ATCC (Manassas, VA). High glucose Dulbecco’s modified Eagle’s medium (DMEM) and Opti-MEM 1 × reduced serum media were purchased from Gibco (Billings, Montana). Ultraculture serum free media was purchased from Lonza (Hayward, CA). Fetal Bovine Serum (FBS) was purchased from HyClone Laboratories (Logan, UT). Polyethylenimine (PEI, Product No. 18978) was purchased from Millipore Sigma. Recombinant Gaussia princeps luciferase (CAT #321-100) was purchased from Nanolight Technologies (Pinetop, AZ). ExoQuick TC was purchased from System Biosciences (Palo Alto, CA). Brefeldin A (BFA) was purchased from eBioscience (San Diego, CA). Pierce™ Gaussia Luciferase Glow Assay Kit was purchased from Thermo Scientific (Waltham, MA).
8
Multiparametric Flow Cytometry of Immune Cells
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For cell surface staining, cells were incubated with anti-mouse FITC-CD11b (Biolegend), APC-CD11c (Biolegend), PE-CD80 (Biolegend), PE-CD86 (Biolegend), and PE-MHCII (Biolegend) after FcR blocking. Isotype-matched mouse immunoglobulin served as control. For intracellular cytokine staining, splenocytes were plated into a 12-well culture plate at a density of 4 × 106/mL and stimulated with 10 μg/mL CAIX protein at 37°C and 5% CO2 for 72 h. These cells were also stimulated with 500 ng/mL Ionomycin (Sigma-Aldrich) and 50 ng/mL PMA (Sigma-Aldrich) plus 5 ng/mL Brefeldin A (BFA, eBioscience) for the last 5 h. Cell surface staining was performed on the stimulated splenocytes or isolated TILs with anti-mouse PerCP-Cy5.5-CD8α (BD Pharmingen), and then intracellular staining was performed with anti-mouse APC-IFN-γ (BD Pharmingen), PE-IL-2 (BD Pharmingen), and FITC-TNF-α (BD Pharmingen). FlowJo software (Tree Star Inc) analyzed the sample data acquired from BD FACSCanto II (BD Biosciences) in FACSDiva software (BD Biosciences).
9
Characterizing Treg-Th17 Plasticity in CD4+ T Cells
10
Cytokine Production Assessment in Viral Infection
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Intracellular cytokine staining was used to assess the extent to which IFN-α was produced by different populations, including pDC, myeloid dendritic cells (mDCs), and monocytes, 24 h post-RV16 stimulation. PBMCs (1 × 106 cells/well) were seeded in a 96-U-bottom plate and stimulated with or without RV16 at 37°C with 5% CO2 for 18 h, and further incubated with Brefeldin A (BFA) (eBioscience) for 4 h. Cells were washed with FACs buffer (1% HI-FBS in PBS) (FBS; Bovogen biological, Australia) and incubated with normal goat IgG (Sigma Aldrich, USA) at 4°C for 15 min to block non-specific Fc binding. The cells were then surface stained with CD303-PE, CD14-PerCP, and CD1c-FITC (Miltenyi Biotec Australia) for 30 min at 4°C, then fixed and permeabilized prior to APC conjugated anti-IFN-α (Miltenyi Biotec Australia) intracellular staining for 30 min at 4°C. The cells were then washed twice with the FACs buffer, and finally fixed in 0.5% paraformaldehyde prior to analysis. Approximately 200,000 gated events per sample were collected using LSRFortessaX-20 (BD-Biosciences, USA), and the results were analyzed using the FlowJo Tree Star software (version 7.6.1). Unstimulated background values were subtracted from the data.
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