Splenic DCs (1 × 105) or peritoneal macrophages were fed with either no antigen,1 μM OVA (Invivogen), or 50 pM SIINFEKL in Iscove’s modified DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% fetal bovine serum (FBS). After 3 hours, cells were washed and 1 × 106 splenocytes from OT-I mice were coincubated. Brefeldin A (BioLegend) was included in some of the cultures. OT-I T cell activation was detected by surface staining with anti-CD69 after 4 hours or by intracellular IFN-γ staining after 6 hours in the presence of Brefeldin A. In some experiments, APCs were pretreated with anti-CD86 (1 μg/ml; GL1, BioLegend) and/or anti-CD80 (1 μg/ml; 16–10A1, BioLegend) before coincubation with T cells.
Brefeldin a
Manufactured by BioLegend
Sourced in United States, Germany, China, United Kingdom, Switzerland
Brefeldin A is a fungal metabolite that functions as a reversible inhibitor of protein transport from the endoplasmic reticulum to the Golgi apparatus. It can be used to induce intracellular accumulation of proteins.
Automatically generated - may contain errors
Lab products found in correlation
Ionomycin, by Merck Group (111 mentions)
Monensin, by BioLegend (104 mentions)
Phorbol myristate acetate (pma), by Merck Group (64 mentions)
Ionomycin, by BioLegend (51 mentions)
Cytofix cytoperm kit, by BD (28 mentions)
Lsrfortessa, by BD (28 mentions)
Cytofix cytoperm, by BD (25 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (21 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (18 mentions)
Golgistop, by BD (17 mentions)
Cell activation cocktail, by BioLegend (16 mentions)
Fixation buffer, by BioLegend (15 mentions)
Streptomycin, by Thermo Fisher Scientific (15 mentions)
Penicillin, by Thermo Fisher Scientific (15 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (15 mentions)
Phorbol myristate acetate (pma), by BioLegend (14 mentions)
Lsrii flow cytometer, by BD (12 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (12 mentions)
Ifn γ, by BioLegend (11 mentions)
Permeabilization buffer, by BioLegend (11 mentions)
659 protocols using brefeldin a
1
Antigen Presentation to CD8+ T Cells
2
SARS-CoV-2 Spike, Nucleocapsid, and Membrane Peptide Stimulation
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SARS-CoV-2 Peptivator peptide pools (Miltenyi Biotech) containing 15-mer sequences with 11 amino acids overlap for the immunodominant section of the spike protein, and the full sequence for nucleocapsid protein and membrane protein (Table S1
PBMC were plated in TexMACS medium (Miltenyi Biotech) in a 24-well plate at 5 × 106 cells/ml per well with treatments: negative control, PMA/ionomycin positive control, individual spike, nucleocapsid, and membrane peptide pools, and combined pools (spike + nucleocapsid + membrane [SNM]). SARS-CoV-2 peptide pools were used at (0.3 nmol/ml), and cell activation cocktail (BioLegend) added to the positive control well at (1×). The negative control well contained DMSO/water at the same volume as the peptide wells. Cells were stimulated for a total of 5 h at 37°C, 5% CO2; with Brefeldin A (BioLegend) added at (5 μg/ml) for the final 3 h (Brefeldin A was only used in this analysis, not in any cell selection experiments).
PBMC were plated in TexMACS medium (Miltenyi Biotech) in a 24-well plate at 5 × 106 cells/ml per well with treatments: negative control, PMA/ionomycin positive control, individual spike, nucleocapsid, and membrane peptide pools, and combined pools (spike + nucleocapsid + membrane [SNM]). SARS-CoV-2 peptide pools were used at (0.3 nmol/ml), and cell activation cocktail (BioLegend) added to the positive control well at (1×). The negative control well contained DMSO/water at the same volume as the peptide wells. Cells were stimulated for a total of 5 h at 37°C, 5% CO2; with Brefeldin A (BioLegend) added at (5 μg/ml) for the final 3 h (Brefeldin A was only used in this analysis, not in any cell selection experiments).
3
Staining and Flow Cytometric Analysis of Lymphocytes and Dendritic Cells
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Cells to be stained with lymphocyte antibodies were stimulated with a cell activation cocktail (2 mg/mL) containing phorbol myristate acetate/ionomycin/Brefeldin A (Biolegend-423303, San Diego, CA) for 4 h at 37°C. Cells to be stained with dendritic cell antibodies were stimulated with R848 (1ug/mL) (Invivogen- tlrl-r848, San Diego, CA) and Brefeldin A (1mg/mL) (Biolegend-42060) for 5 h at 37°C. Detailed staining protocol is included in Supplementary methods . Subsequent to staining, single-cell suspensions underwent flow cytometric analysis on a BD FACS Canto (5 samples) or BD FACS Symphony (2 samples) (BD Biosciences, San Jose, CA) and were analyzed with the FlowJo software (BD Biosciences). The intensity of the staining was measured by percent of parent population, and gating was determined by the mean fluorescence intensity (MFI) values. To distinguish between specifically stained cells and background fluorescence, we used appropriate controls, including unstimulated samples and fluorescence-minus-one controls [48 (link)]. We defined this MFI value as a cut-off and considered a cell as positive for a given marker if their MFI exceeded this cut-off value and cells with MFI values below this cut-off value were referred to as cytokine-negative cells. Further data regarding gating process can be found in the Supplementary methods .
4
Stimulation and Intracellular Staining of Isograft Cells
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Isografts were disaggregated as described. For stimulation, PMA (phorbol 12-myristate 13-acetate) (Sigma), ionomycin (Sigma) and Brefeldin A (BioLegend) were added at the following concentrations: PMA 50 ng ml−1, ionomycin 0.709 μg ml−1, Brefeldin A 5 ug ml−1. Cells were plated onto 96-well plates using one well per mouse (2 isografts). The plate was incubated for 3 h at 37 °C then the contents of each well was transferred to a 1.5 ml eppendorf tube for staining. Initial cell surface staining was performed as described above (CD45-PE-Cy7, CD3-APC, CD8-Alexafluor 700) followed by fixation of the cells using 4% formaldehyde in PBS. After fixation, permeabilization buffer (BD Bioscience) was added, cells were spun down and resuspended in a wash of permeabilization buffer then spun down again before resuspension with antibodies against IFNγ or a control IgG (IFNγ-PE or IgG-PE). Intracellular staining was carried out for 1 h on ice in the dark. Cells were washed and transferred to FACS tubes for analysis using a BD LSRII cytometer (Becton Dixon) and FlowJo software (Tree Star). Additional information about antibodies can be found in Supplementary Table 1 .
5
Multiparametric Analysis of Cytokine Production
6
Cytokine Production Assay in Leukocytes
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As we described previously in our study20 (link), isolated leukocytes (2 × 106 cells/mL) were resuspended in complete medium [RPMI 1640 medium-10% fetal bovine serum containing penicillin (200 μg/mL), streptomycin (200 U/mL), 4 mmol/L L-glutamine, and 5 × 10−5 mol/L 2-mercaptoethanol; all from Gibco, Carlsbad, CA, USA] and stimulated with lipopolysaccharide (LPS; 10 μg/mL; Escherichia coli serotype 0111: B4; Sigma-Aldrich, St louis, Mo, USA), phorbol 12 myristate 13-acetate (PMA; 50 ng/mL, Sigma-Aldrich), ionomycin (500 ng/mL, Sigma-Aldrich), and brefeldin A (3 μM, BioLegend) for IL-10 and PMA, ionomaycin, and brefeldin A for IL-17 for 5 h at 37℃. Dead cells were detected using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen) before cell surface staining. Stained cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s instructions and stained with PE or APC -conjugated mouse anti-IL-10 mAb and APC-conjugated mouse anti-IL-17A mAb.
7
Comprehensive Immune Cell Profiling
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Single cell suspensions were analyzed by flow cytometry using the following antibodies: CD3 (17A2), CD4 (RM4-4), CD8 (53–6.7), I-Ab (AF6-120.1), CD44 (IM7), CD62L (MEL14), CD86 (GL-1), CD11b (M1/70), CD11c (N418), CD25 (PC61), NK1.1 (PK136), CD27 (LG.3A10), CD90.2 (53-2.1), Ki67 (16A8), CD127 (A7R34), CD49b (DX5), KLRG1 (2F1/KLRG1) and SIRPα (P84). Zombie Aqua fixable viability dye (Cat. No. 423102, BioLegend, San Diego (CA), USA) was used to exclude all non-viable cells from the analysis. Anti-CD16/CD32 was added to antibody mix to block non-specific mAb binding. FACS buffer containing 0.5% BSA and 0.02% sodium azide in PBS without Ca2+ and Mg2+ was used for all the washing steps.
To measure intracellular cytokines, splenocytes or aorta digested cells were stimulated with cell activation cocktail containing PMA, ionomycin and BrefeldinA (BrefA) (Cat No. 423303, Biolegend) or given Brefeldin A (Biolegend) alone for 4 hours at 37 °C. Cells were fixed and permeabilized using a fixation and permeabilization kit (Cat. No. 88-8824-00, eBioscience) and subsequently stained with IFN-γ (XMG1.2) or Granzyme B (QA16A02). FACS analysis was performed on a DxP11 flow cytometer (Cytek, Fremont (CA), USA) and the data were analyzed using FlowJo software (FlowJo LLC, Ashland (OR), USA).
To measure intracellular cytokines, splenocytes or aorta digested cells were stimulated with cell activation cocktail containing PMA, ionomycin and BrefeldinA (BrefA) (Cat No. 423303, Biolegend) or given Brefeldin A (Biolegend) alone for 4 hours at 37 °C. Cells were fixed and permeabilized using a fixation and permeabilization kit (Cat. No. 88-8824-00, eBioscience) and subsequently stained with IFN-γ (XMG1.2) or Granzyme B (QA16A02). FACS analysis was performed on a DxP11 flow cytometer (Cytek, Fremont (CA), USA) and the data were analyzed using FlowJo software (FlowJo LLC, Ashland (OR), USA).
8
Cytokine-stimulated IFN-γ Production in NK Cells
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For cytokine stimulation experiments, 2 × 10 4 mouse NK cells were stimulated for 4 h in CR-10 (RPMI 1640 + 25 mM HEPES (Gibco-15630-080) + 10% FBS, 1% l-glutamine, 1% 200 mM sodium pyruvate, 1% MEM-NEAA, 1% penicillin-streptomycin, 0.5% sodium bicarbonate and 0.01% 55 mM 2-mercaptoethanol), brefeldin A (1:1,000 dilution; BioLegend) and monensin (2 μM; BioLegend) with or without recombinant mouse IL-15 (50 ng ml -1 ; PeproTech), mouse IL-12 (20 ng ml -1 ; PeproTech) and/or recombinant mouse IL-18 (10 ng ml -1 ; BioLegend, 767002). Cells were cultured in CR-10 medium alone as a negative control (no treatment). The absolute number of IFN-γ-producing NK cells were determined by acquiring and counting individual cells with an Attune NxT after intracellular flow cytometry staining to determine cell count of IFN-γ + of the 2 × 10 4 cells plated per condition. For plate-bound antibody stimulation experiments, 2 × 10 4 isolated NK cells for each condition were stimulated with 4 mg ml -1 precoated antibody against NK1.1 (PK136) for 4 h in complete medium containing brefeldin A (1:1,000 dilution; BioLegend) and monensin (2 μM; BioLegend). Cells were cultured in medium alone as a negative control (no treatment).
9
Cytokine Expression Profiling of Activated T Cells
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Activated Jurkat T cells were treated by brefeldin A for 6 hours before collecting, fixed in 4% paraformaldehyde for 20 min at room temperature, permeabilized with intracellular staining wash buffer (Biolegend), cells were blocked with PBS containing 5% PBS, IL-2, TNF-α, IFN-γ were then stained.
Splenocytes were subjected to erythrocyte lysis buffer and prepared by passing spleen tissue through a 70‐μm cell strainer, and then treated by cell stimulation cocktail (eBioscience) and brefeldin A (Biolegend) for 6 hours. After stained with zombie aqua and the surface markers, splenocytes were fixed in fixation buffer (Biolegend) for 20 min at room temperature, permeabilized with intracellular staining wash buffer, blocked with anti-CD16/32, and then stained with anti-IFN-γ, granzyme B, and perforin.
Appropriate isotype controls were used, and compensation was made according to single staining. Percentage of positive cells and MFI were analyzed with FlowJo software (Tree Star).
Splenocytes were subjected to erythrocyte lysis buffer and prepared by passing spleen tissue through a 70‐μm cell strainer, and then treated by cell stimulation cocktail (eBioscience) and brefeldin A (Biolegend) for 6 hours. After stained with zombie aqua and the surface markers, splenocytes were fixed in fixation buffer (Biolegend) for 20 min at room temperature, permeabilized with intracellular staining wash buffer, blocked with anti-CD16/32, and then stained with anti-IFN-γ, granzyme B, and perforin.
Appropriate isotype controls were used, and compensation was made according to single staining. Percentage of positive cells and MFI were analyzed with FlowJo software (Tree Star).
10
Intracellular Cytokine Analysis of Murine Skin Cells
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Whole skin cell suspensions from the ears of WT or p32cKO mice were cultured in cRPMI in the presence of mouse IL-23 (15 ng/mL, BioLegend), IL-1β (30 ng/mL, PeproTech), or IL-23 and IL-1β for 72 h. Brefeldin A (5 µg/mL, BioLegend) was added 2–3 h before intracellular staining and flow cytometry.
Whole skin cell suspensions from the ears of WT or p32cKO mice treated with IMQ for 4 consecutive days were re-stimulated with phorbol myristate acetate (PMA, 1 µg/mL, Wako Pure Chemical Industries) and ionomycin (0.1 µg/mL, Wako Pure Chemical Industries) in the presence of Brefeldin A (5 µg/mL, BioLegend) for 2–3 h. They were then stained by the intracellular staining procedure as described to determine IL-17A expression (35 (link)).
Whole skin cell suspensions from the ears of WT or p32cKO mice treated with IMQ for 4 consecutive days were re-stimulated with phorbol myristate acetate (PMA, 1 µg/mL, Wako Pure Chemical Industries) and ionomycin (0.1 µg/mL, Wako Pure Chemical Industries) in the presence of Brefeldin A (5 µg/mL, BioLegend) for 2–3 h. They were then stained by the intracellular staining procedure as described to determine IL-17A expression (35 (link)).
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