The splenocyte-derived cytokines were measured using intracellular cytokine staining (ICS), as described in the Supplementary Materials. Briefly, splenocytes were collected 3 weeks after the third immunization and subsequently pulsed with an anti-CD28 agonist antibody (BD Biosciences, USA) in combination with either RBD or a set of 14-mer peptides covering the RBD region in overlap (NR-52402, BEI Resources, USA). After 1 h, the cells were treated with brefeldin A (BD Biosciences, USA) and incubated for 12 h at 37 °C and 5 % CO2. Non-pulsed cells served as the negative control. Extracellular staining was performed using Live/Dead Fixable Aqua Dead, anti-CD3 BV786 (BioLegend, USA), anti-CD8 APC (BioLegend, USA), and anti-CD4 BV605 (BioLegend, USA). After washing, the fixed and permeabilized cells were intracellularly stained with anti-IFNγ PE (BioLegend, USA), anti-IL-2 BV421 (BioScience), and anti-TNFα Pe-Cy7 (BD Bioscience, USA). The cells were subsequently analyzed using an LSR Fortessa flow cytometer (BD Biosciences, USA).
Anti tnfα pecy7
Manufactured by BD
Sourced in United States
Anti-TNFα-PECy7 is a fluorescent-labeled antibody that binds to Tumor Necrosis Factor alpha (TNFα). It is used for the detection and analysis of TNFα in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Brefeldin a, by BD (9 mentions)
Anti cd28, by BD (4 mentions)
Monensin, by BD (4 mentions)
Anti cd49d, by BD (4 mentions)
Anti ifn γ alexa 700, by BD (4 mentions)
Anti cd107a fitc, by BD (4 mentions)
Anti il 2 apc, by BD (4 mentions)
Anti ifn γ apc, by BD (4 mentions)
Lsrfortessa flow cytometer, by BD (3 mentions)
Anti cd8 pe, by BD (3 mentions)
Live dead cell dye, by Thermo Fisher Scientific (3 mentions)
Anti cd3 alexa 780, by Thermo Fisher Scientific (3 mentions)
Anti granzyme b v450, by BD (3 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Golgistop, by BD (2 mentions)
Anti cd8 v500, by BD (2 mentions)
Anti cd3 percp, by BD (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Anti ifn γ pe, by BD (2 mentions)
Anti il 2 apc cy7, by BD (2 mentions)
18 protocols using anti tnfα pecy7
1
Intracellular Cytokine Profiling of T-Cell Response
2
Cytokine Production Assay Protocol
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During the last 4 or 16 hours of culture depending upon the experiment, Brefeldin A (3 μg/ml) and Golgistop (1/2000 final dilution; both from BD Biosciences, Mountain View, CA) were added to block cytokine secretion. For diluted whole blood cultures, red cells were lysed with 5 volumes of 1x PharmLyse solution (BD Biosciences, Oxford, UK) for 10 minutes at room temperature. Labeling of dead cells, fixation and permeabilization were performed as previously described [24 (link)]. Depending upon the experiment, cells were surface stained with anti-CD4-APC-H7 (BD Biosciences), anti-CD19-efluor450 and anti-CD14-efluor450 (eBiosciences) for 30 minutes at 4°C, or, following permeabilisation, with anti-CD3-Horizon-V500, anti-IL-2-FITC, anti-TNFα-PE-Cy7 (BD Biosciences), anti-IL-17-efluor660, and anti-IFNγ-PerCP-Cy5.5 (Biolegend) for 30 minutes at room temperature. Cells were finally resuspended in 250 μL 1% paraformaldehyde (Sigma, UK) and filtered prior to acquisition on a FACS Canto II flow cytometer or an LSRII flow cytometer (BD Biosciences). Compensation was performed using tubes of CompBeads (BD Biosciences) individually stained with each fluorophor and compensation matrices were calculated with FACSdiva. Data were analyzed using FlowJo software version 9 (Treestar, Ashland, OR). Gating strategy and an example of raw flow cytometry data is shown in S1 Fig .
3
Polyfunctional T-cell Profiling by Flow Cytometry
4
Multiparametric Flow Cytometry for Immune Profiling
5
Polyfunctional T-cell Immune Response Analysis
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Flow cytometry based ICS assay was done as previously described [35 ,49 (link),50 (link)]. In brief, 106 PBMCs were pulsed with peptide at 10uM in the presence of co-stimulatory antibodies (anti-CD28 and anti-CD49D) and anti-CD107a-FITC (all from BD Biosciences) for 2 hrs at 37°C. Monensin and brefeldin A (both from BD Biosciences) were then added and the cultures incubated for an additional 12 hrs. Next day, the cells were labeled with LIVE/DEAD cell dye (Invitrogen) and surface stained with anti-CD3-Pac Blue, anti-CD8-V500, and anti-CD4-Alexa 780 (both from BD Biosciences). The cells were permeabilized and labeled with anti-IFN-γ-Alexa 700, anti-IL-2-APC, anti-TNFα-PECy7, and anti-Granzyme A-PE (all from BD Biosciences). CD3 events greater than 100,000 were acquired on an LSR II (BD Immunocytometry Systems), and data were analyzed using FlowJo (version 9.6.4; TreeStar). Polyfunctionality analysis was performed using Boolean gating and SPICE and Pestle software (version 5.1; NIAID).
6
Dendritic Cell Maturation and T-Cell Activation
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To determine maturation status, DCs were harvested two days after addition of peptides and maturation factor LPS. Cells were stained in FACS buffer (PBS (GIBCO) containing 0.5% BSA (Sigma) and 0.5 mM EDTA (ICN Biomedicals)) for 30 minutes at 4°C with either one of two panels that contained the following maturation markers: anti-CD80-FITC, anti-CD14-PE, anti-DC-SIGN-APC, anti-HLA-DR-Pacific Blue and Live/dead-AmCyan (Invitrogen) (panel 1) or anti-CD83-FITC, anti-CD40-PE, (BD Biosciences), anti-PD-L1-APC (eBioscience), anti-CD86-Pacific Blue (BioLegend) (panel 2). Live/dead-AmCyan (Invitrogen) was included in both panels. For analysis of the co-culture, the following markers were used: anti-CD8-FITC (Sanquin), anti-CD3-PerCP, anti-TNFα-PE-Cy7, anti-IFN-γ-APC (BD Biosciences), anti-CD4-Pacific Blue (eBioscience) and Live/dead-AmCyan (Invitrogen). Four hours prior to staining, Brefeldin A (BD Biosciences) was added to the culture; then cells were stained using the Cytofix/Cytoperm kit from BD Biosciences according to manufacturer’s recommendations. Cells were measured using a FACS Canto II (BD Biosciences) and results were analyzed using FlowJo version 9.7.5 software. First, lymphocytes were gated, followed by gating of live cells, then CD3+ cells and finally CD8+ or CD4+ cells were placed in a quadrant with TNF-α+ or IFN-γ+ cells.
7
Flow Cytometry of Immune Markers
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The following antibodies were used for flow cytometry: anti-CD4 PerCP/Cy5.5 (Clone GK1.5, BioLegend), anti-CD3 APC-Cy7 (Clone 145-2C11, BD Biosciences), anti-TNF-α PE-Cy7 (Clone MP6-XT22, BD Biosciences), anti-IFN-γ AF700 (Clone XMG1.2, BD Biosciences), anti-IL-2 APC (Clone JES6-5H4, BD Biosciences).
8
Multi-Color ICS for Cytokine Analysis
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Multi-color ICS was performed as previously reported (29 (link)). Briefly, 1 × 106 freshly isolated mouse splenocytes were incubated with peptide pool s for 2 h. Brefeldin A (eBioscience) was added to block cytokine secretion for 8 h. Cells were stained with antibody cocktails (anti-CD3-PerCP, anti-CD4-APC and anti-CD8-FITC) (BD Bioscience) for 30 min at room temperature, permeabilized using Cytofix/Cytoperm buffer (BD Bioscience), and stained with anti-IFN-γ-PE, anti-TNF-α-PE-Cy7, and anti-IL-2-APC-Cy7 (BD Bioscience). Samples were subjected to flow cytometric analysis using the FACSArial instrument and data were processed with FlowJo 7.6 software (Tree Star, Inc.).
9
Comprehensive Multiparametric Phenotyping of PBMCs
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After stimulation, PBMCs were labeled with viability marker LIVE/DEAD Near-IR fluorescent reactive dye (Thermo Fisher) for 30 min and subsequently stained for 20 min with the following surface markers: anti-CD3-PerCP (BioLegend), anti-CD4-BV786, anti-CD8-BV510, anti-CD27-BV605, anti-CD38-PE, and anti-HLA-DR-BV421 (BD Bioscience). Cells were then fixed and permeabilized with the Foxp3 Transcription Factor Staining Buffer Set (Thermo Fisher) and stained for 30 min with intracellular markers: anti-IFNγ-APC, anti-TNFα-PE-Cy7 and anti-Ki-67-FITC (BD Bioscience). The complete list of antibodies, conjugated proteins and dilutions can be found in Supplementary Table 1 . All incubation processes were performed at room temperature in darkness. Fluorescence Minus One (FMO) controls of the four analyzed cell markers (CD27, CD38, HLA-DR, and Ki-67) were included in each run to accurately distinguish negative from positive populations. Samples were resuspended in 100 μl of PBS-0.1%BSA (Bovine Serum Albumin, Sigma Aldrich) and acquired in a BD LSRFortessa flow cytometer (BD Bioscience) using FACSDiva software (BD Biosciences) with compensated parameters.
10
T-cell Cytokine Profiling in PBMC
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PBMC were incubated with medium in presence of CD28 (1 μg/ml) and CD49d (1 μg/ml) mAbs in a 200 μl final volume at 37 °C, 5% CO2 for one hour, followed by five-hour incubation in the presence of brefeldin A (GolgiPlug, BD). After a total of six hours incubation, cells were for surface and intracellular staining. In surface staining, cells were incubated with CD3-PBCD4-BV510, CD8-AF700, Vδ2-FITC solutions for 30 min at 4 °C without Ag stimulation in vitro. And then, cells were washed, fixed, permeabilized with FACS perm buffer (PBS supplemented with 0.5% Bovine Serum Albumin-BSA, 0.5% of saponin and 0.1% sodium azide) and incubated in solutions with anti-IFN-γ-BV711, anti-TNF-α-PE-CY7 and anti-IL-17A-PE (BD Biosciences - San Jose, CA, USA) for 30 min at 4 °C. The preparations, then maintained in 200 μL of FACS fix solution until acquisition in a Becton Dickinson FACS calibur instrument (Additional file 5 ). Isotype IgG was used as negative controls for staining cytokines or surface markers.
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