Thawed PBMC were washed twice with complete culture medium (RPMI 1640 supplemented with 10% fetal bovine serum and 1% each of l ‐glutamine, sodium pyruvate, nonessential amino acids, antibiotics, 0.1 M HEPES, 55 μM β‐mercaptoethanol) plus 0.02 mg/ml DNAse. PBMCs were stimulated for 4 h at 37°C in a 5% CO2 atmosphere with PMA (100 ng/ml) and Ionomycin (1 μg/ml) in complete culture medium. For each sample, at least 2 million cells were left unstimulated as negative control, and 2 million cells were stimulated. All samples were incubated with a protein transport inhibitor containing brefeldin A (Golgi Plug, Becton Dickinson). After stimulation, cells were stained with LIVE‐DEAD Aqua (Thermo Fisher Scientific) and surface mAbs recognizing HLA‐DR‐PE‐Cy7, CD14‐APC, and CD16‐BV421 (BioLegend, San Diego, CA, USA). Cells were washed with stain buffer, fixed, and permeabilized with the cytofix/cytoperm buffer set (Becton Dickinson) for cytokine detection. Then, cells were stained with previously titrated mAbs recognizing IFN‐γ‐FITC and TNF‐BV605 (all mAbs from BioLegend). Samples were acquired on Attune NxT acoustic cytometer (Thermo Fisher).
Tnf bv605
Manufactured by BioLegend
TNF-BV605 is a fluorochrome-conjugated antibody that binds to the tumor necrosis factor (TNF) protein. It can be used for the detection and quantification of TNF in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Golgiplug, by BD (3 mentions)
Live dead aqua, by Thermo Fisher Scientific (3 mentions)
Cytofix cytoperm buffer set, by BD (3 mentions)
Attune nxt acoustic cytometer, by Thermo Fisher Scientific (3 mentions)
Cd8 apc cy7, by BioLegend (2 mentions)
Ifn γ fitc, by BioLegend (2 mentions)
Il 17 bv421, by BioLegend (2 mentions)
Cd14 apc, by BioLegend (1 mentions)
Cd16 bv421, by BioLegend (1 mentions)
Il 17a pe, by BioLegend (1 mentions)
Cd3 pe cy7, by BioLegend (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Golgistop, by BD (1 mentions)
Live dead efluor780, by Thermo Fisher Scientific (1 mentions)
Cd3 pe cy7 ucht1, by BioLegend (1 mentions)
Epr9342 b, by Abcam (1 mentions)
Ifn γ pacific blue, by BioLegend (1 mentions)
Foxp3 staining buffer, by BioLegend (1 mentions)
Epr25a, by Abcam (1 mentions)
5 protocols using tnf bv605
1
Multiparametric Immune Cell Profiling
2
Multiparametric Intracellular Cytokine Analysis
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For intracellular cytokine staining, CD4+ T cells or CD4+ T cell/monocyte cocultures were stimulated for 3 h in the presence of PMA (50 ng/ml; Sigma-Aldrich), ionomycin (750 ng/ml; Sigma-Aldrich), and GolgiStop (as per the manufacturer’s instructions; BD Biosciences). Cells were washed and stained with CD3-PE Cy7 (UCHT1; BioLegend) and LIVE/DEAD efluor 780 (Thermo Fisher Scientific). Cells were then fixed in 2% PFA and permeabilized with 0.5% saponin (Thermo Fisher Scientific). Cells were subsequently stained for the following cytokines: IL-10–Alexa Fluor 488 (JES3-9D7; BioLegend), IL-17A–PE (BL168; BioLegend), IFN-γ–Pacific blue (4S.B3; BioLegend), and TNF–allophycocyanin (MAb11; BioLegend).
For intranuclear staining of IKZF3, cells were fixed and permeabilized with FOXP3 staining buffer (BioLegend) for 15 min at room temperature before being stained for CD3-PE Cy7, IL-10–Alexa Fluor 488, IL-17A–PE, IFN-γ–Pacific blue, TNF-BV605 (MAb11; BioLegend), and either IKZF3-Alexa Fluor 647 (EPR9342[B]; Abcam) or isotype control (EPR25A; Abcam) for 30 min. Standard gating strategy for intracellular cytokine staining is shown inSupplemental Fig. 1A–C .
For intranuclear staining of IKZF3, cells were fixed and permeabilized with FOXP3 staining buffer (BioLegend) for 15 min at room temperature before being stained for CD3-PE Cy7, IL-10–Alexa Fluor 488, IL-17A–PE, IFN-γ–Pacific blue, TNF-BV605 (MAb11; BioLegend), and either IKZF3-Alexa Fluor 647 (EPR9342[B]; Abcam) or isotype control (EPR25A; Abcam) for 30 min. Standard gating strategy for intracellular cytokine staining is shown in
3
Cytokine Production Profiling of Activated T Cells
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For functional assays on cytokine production by T cells, thawed isolated PBMCs were stimulated for 16 h at 37 °C in a 5% CO2 atmosphere with anti-CD3/CD28 (1 μg/mL) in complete culture medium (RPMI 1640 supplemented with 10% fetal bovine serum and 1% each of l-glutamine, sodium pyruvate, nonessential amino acids, antibiotics, 0.1 M HEPES, 55 μM β-mercaptoethanol). For each sample, at least 2 million cells were left unstimulated as negative control, and 2 million cells were stimulated. All samples were incubated with a protein transport inhibitor containing brefeldin A (Golgi Plug, Becton Dickinson) and previously titrated concentration of CD107a-PE. After stimulation, cells were stained with LIVE-DEAD Aqua (ThermoFisher Scientific) and surface mAbs recognizing CD3 PE- Cy5, CD4 AF700, and CD8 APC-Cy7 (Biolegend, San Diego, CA, USA). Cells were washed with stain buffer, and fixed and permeabilized with the cytofix/cytoperm buffer set (Becton Dickinson) for cytokine detection. Cells were next stained with previously titrated mAbs recognizing IL-17 BV421, TNF BV605, IFN-γ FITC, IL-2 APC, or granzyme-B BV421 (all mAbs from Biolegend). Then, a minimum of 100,000 cells per sample were acquired on a Attune NxT acoustic cytometer (ThermoFisher).
4
Comprehensive Lymphocyte Immunophenotyping
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The following antibodies were used for phenotyping and functional evaluation of lymphocytes in this study: CD107a-BV421 (Clone H4A3, Biolegend), CD4-PECy5.5 (Clone S3.5, Invitrogen), CD8-BV570 (Clone RPA-T8, Biolegend), CD14-BV650 (Clone M5E2, Biolegend), CD20-BV650 (Clone 2H7, Biolegend), CD16-BV650 (Clone 3G8, Biolegend), CD28-ECD (Clone CD28.2, Beckman Coulter), CD95-PECy5 (Clone Dx2, BD Biosciences), CD38-PE (Clone OKT10, NIH NHP Reagent Resource, University of Massachusetts, Boston, MA), CCR7- BV711 (Clone G043H7, Biolegend). CD3-APCCy7 (Clone SP34-2, BD Biosciences), granzyme B-AF700 (Clone GB11, BD Biosciences), T-bet-PECy7 (Clone 4B10, eBioscience), Ki67-FITC (Clone B56, BD Bioscience) or Ki67-BV786 (Clone B56, BD Bioscience), CD69-APC (Clone FN50, BD Bioscience) or CD69-BV605 (Clone FN50, Biolegend), Perforin-FITC (Clone pf344, MabTech), TNF-BV605 (Clone MAb11, Biolegend), IFNg-BV785 (Clone 4S.B3, Biolegend), Live Dead Fixable Aqua Dead Cell Stain (Molecular Probes).
5
Cytokine Production Profiling of Activated T Cells
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For functional assays on cytokine production by T cells, thawed isolated PBMCs were stimulated for 16 h at 37 °C in a 5% CO2 atmosphere with anti-CD3/CD28 (1 μg/mL) in complete culture medium (RPMI 1640 supplemented with 10% fetal bovine serum and 1% each of l -glutamine, sodium pyruvate, nonessential amino acids, antibiotics, 0.1 M HEPES, and 55 μM β-mercaptoethanol). For each sample, at least 2 million cells were left unstimulated as negative control, and 2 million cells were stimulated. All samples were incubated with a protein-transport inhibitor containing brefeldin A (Golgi Plug, Becton Dickinson) and previously titrated concentration of CD107a-PE. After stimulation, cells were stained with LIVE-DEAD Aqua (ThermoFisher Scientific) and surface mAbs recognizing CD4 AF700, and CD8 APC-Cy7 (Biolegend, San Diego, CA, USA). Cells were washed with stain buffer and fixed and permeabilized with the cytofix/cytoperm buffer set (Becton Dickinson) for cytokine detection. Cells were next stained with previously titrated mAbs recognizing CD3 PE-Cy5, IL-17 BV421, TNF BV605, IFN-γ FITC, IL-4 APC, or granzyme-B BV421 (all mAbs from Biolegend). Then, a minimum of 100,000 cells per sample were acquired on a Attune NxT acoustic cytometer (ThermoFisher)53 (link). FCS data were acquired in list mode by Attune nxT Software v4.2. mAbs used are listed in Sup Data 6 .
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