Surface staining was performed using the following antibodies: CD117 (c-Kit)-APC or APC/Cy7, FcεRIα-PE, CD11b (Mac-1)-Pacific Blue (all from Biolegend) and Ly-6G (Gr-1)-PE-Cyanine7 (eBioscience). All antibodies for surface staining were used at a dilution of 1:200. For intracellular cytokine staining, cells were stimulated with 1 μg/ml IgE–anti-DNP antibody and 0.2 μg/mL HSA-DNP antigen (both from Sigma) for 3.5 h, with the addition of brefeldin A in the last two hours of stimulation, as previously described3 (link),4 (link). The following antibodies were used: IL-6-PE or APC, TNF-α-PE/Cy7 (both from Biolegend) and IL-13-PE (eBioscience). All antibodies used in this study are listed in Supplementary Table 1 . Flow-cytometry data were collected on a FACS Symphony A5 or Fortessa (BD Biosciences) and analyzed by FlowJo version 10 (BD Biosciences).
Tnf α pe cy7
Manufactured by BioLegend
Sourced in United States
The TNF-α-PE/Cy7 is a fluorescent-labeled antibody that binds to the human tumor necrosis factor alpha (TNF-α) protein. It is commonly used in flow cytometry applications to detect and quantify the expression of TNF-α in cell samples.
Automatically generated - may contain errors
Lab products found in correlation
Fcεriα pe, by BioLegend (2 mentions)
Il 13 pe, by Thermo Fisher Scientific (2 mentions)
Pd1 pe cy7, by BioLegend (2 mentions)
Klrg1 fitc, by BioLegend (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Il 6 pe, by BioLegend (2 mentions)
Anti dnp ige antibody, by Merck Group (1 mentions)
Facs symphony a5, by BD (1 mentions)
Fortessa, by BD (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Cd45 af700, by BioLegend (1 mentions)
Facs symphony cytometer, by BD (1 mentions)
Cd8α buv805, by BD (1 mentions)
Nk1.1 bv785, by BioLegend (1 mentions)
Cd11b bv711, by BioLegend (1 mentions)
Ly6g bv605, by BioLegend (1 mentions)
F4 80 bv785, by BioLegend (1 mentions)
B220 bv785, by BioLegend (1 mentions)
Fas pe cy7, by BioLegend (1 mentions)
Cd38 percp cy5, by BioLegend (1 mentions)
14 protocols using tnf α pe cy7
1
Flow Cytometric Immunophenotyping of Mast Cells
2
Comprehensive Immune Cell Profiling
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Cells from each tissue were resuspended in PBE 1x (PBS supplemented with 0.5% BSA + 1 mM EDTA) and incubated for 30 min on ice with fluorescently labeled antibodies: CD45-AF700 (BioLegend Cat. No. 103127), CD4-BUV495 (BD Biosciences Cat. No. 565974), CD8α-BUV805 (BD Biosciences Cat. No. 612898), TCRβ-BUV395 (BD Biosciences Cat. No. 742485), NK1.1-BV785 (BioLegend Cat. No. 108749), CD11b-BV711 (BioLegend Cat. No. 101241), CD11c-BUV496 (BD Biosciences Cat. No. 750483), I-A/I-E-BUV395 (BD Biosciences Cat. No. 743876), Ly6G-BV605 (BioLegend Cat. No. 127639), F4/80-BV785 (BioLegend Cat. No. 123141), IFN-δ-PerCP-Cy5.5 (BD Biosciences Cat. No. 560660), TNF-α-PE-Cy7 (BioLegend Cat. No. 506323), IL-17α-BV421 (BioLegend Cat. No. 506925), B220-BV785 (BioLegend Cat. No. 103245), FAS-PE-Cy7 (BioLegend Cat. No. 152617), CD38- PerCP-Cy5.5 (BioLegend Cat. No. 102721) and CD138-BV650 (BioLegend Cat. No. 142517). WA-1 RBD and Omicron RBD biotinylated were purchased from Sino Biological and conjugated with streptavidin. LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, L-34965, was purchased from Life Technologies. Cells were filtered and washed with PBE 1x again before analysis on BD FACS Symphony cytometer. Analyses were performed using FlowJo v. 10 software.
3
Quantifying LCMV-Specific CD8+ T Cell Responses
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Splenocytes were harvested from mice 7 days post-infection. Harvested splenocytes were incubated for 5 h in RPMI-1640 medium containing µg/mL GP33 (Genescript, Piscataway, NJ, USA) and 2 µg/mL brefeldin A (Sigma, St. Louis, MO, USA). Cells were stained with anti-mouse CD3-APC Cy7, CD8-PE, CD45.1-PerCP Cy5.5, and LCMV GP33 Tetramer-APC and permeabilized using Foxp3/Transcription Factor Staining Buffer Kit according to the manufacturer’s protocol. After permeabilization, cytokines were detected using anti-mouse IFN-γ-FITC and TNF-α-PE Cy7 (Biolegend, San Diego, CA, USA).
4
Multicolor Flow Cytometry Panel
5
Multiparametric Flow Cytometry Staining
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Staining for surface markers was performed by incubating cells with antibody at 1:200 dilutions in fluorescence-activated cell sorting buffer (0.1% BSA in PBS) for 30 min at 4 °C. For intracellular cytokine staining of IFNγ and TNFα, surface markers were stained before fixation/permeabilization (BD Cytofix/Cytoperm Kit, #554714; BD Biosciences) (40 (link)). Flow antibodies were purchased from BioLegend with the information as follows: CD8a-BV786 (#563332), CD11c-BV786 (#563735), PD1-PE/Cy7 (#109109), TIM3-APC (#119705), KLRG1-FITC (#138409), LAG3-PerCP/Cy5.5 (#125211), TNFα-PE/Cy7 (#506323), and Granzyme B-Alexa 700 (#372221). Samples were acquired on BD LSRFortessa flow cytometer and analyzed with FlowJo software (https://www.flowjo.com/solutions/flowjo ) (Tree Star) unless otherwise stated.
6
T Cell Functionality via Cytokine Staining
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The functionality of pMHC-specific T cells was tested by combining intracellular cytokine staining (ICS) with a DNA barcode-based pMHC multimer staining, as previously described in Bentzen et al.20 Briefly, patient-derived PBMCs were stimulated with either pre-stimulated IFNγ (PreproTech, Cat#300-02) breast cancer cell lines at a 10:1 ratio (PBMCs:cell lines), a pool of peptides with known reactivity in the specific patient or a leucocyte activation cocktail (LAC, BD, Cat#550583) positive control for 4 h at 37°C. After stimulation, cells were stained with barcoded MHC multimers, followed by staining with extracellular surface antibodies: CD3-FITC (BD, Cat#345763), CD8-BV480, and dead cell marker LIVE/DEAD Fixable Near-IR. Hereafter cells were permeabilized with the Intracellular Fix and Perm kit (eBioscience, Cat#88-8824-00), stained with intracellular antibodies TNFα-PE-Cy7 (BioLegend, Cat#502930) and IFNγ-APC (BD, Cat#341117), and fixated in 1% PFA until analysis.
7
Multicolor Flow Cytometry Immunophenotyping
8
Multiparameter Flow Cytometry Analysis
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Cell surface and intracellular molecular expressions were evaluated by flow cytometry using CytoFLEX (Beckman Coulter, U.S.A.). Fluorescein-conjugated mouse anti-human antibodies were used, including CD3-FITC/PE, CD4-FITC/APC, IFN-γ-PE-CY7/PE, TNF-α-PE-CY7, IL-4-PE/PE-CY7/BV421, IL-5-BV421, IL-13-BV421, T-bet-PE-CY7 and GATA-3-BV421 (Biolegend, UK). Anti-human antibodies NR2F2(Abcam, UK) were binding with fluorescent agent APC (Biolegend, UK) according to the manufacturer’s introduction. For cell-surface staining, single-cell suspensions were stained on ice for 30 min in PBS with 1% fetal bovine serum (FBS). For intracellular staining, cells were fixed and permeabilized using the Fix/Perm kit (Biolegend, UK). To detect intracellular cytokines, CD4+T cells were stimulated for 4 h with phorbol 12-myristate 13-acetate (PMA; 1 μg/mL; Sigma) and ionomycin (1 μg/mL; Sigma), and 4 h with GolgiStop (1 μL/mL; BD Biosciences) in a round-bottom 96-well plate. Thereafter, cells were harvested, stained for surface expression, and then fixed and permeabilized for intracellular staining. Flow cytometry data was analyzed using FlowJo software (BD, UK) and CytoExpert software (Beckman Coulter, U.S.A.).
9
Comprehensive Immune Cell Profiling
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Antibodies/dyes and dilutions used were: viability dye live/dead fixable-violet (L34955, Invitrogen, 1:1250), CD3-eFluor450 (48-0038, eBioscience, 1:100), CD3-PECy7 (25-0038, eBioscience, 1:100), CD4-VioGreen (130-096-900, Miltenyi Biotech, 1:50), CD8-VioGreen (130-098-062, Miltenyi Biotech, 1:50), CD8-V450 (560347, BD, 1:50), CD8-PerCP.Cy5.5/PerCP (301032, Biolegend, 1:100), CD38-APC (555462, BD, 1:50), CD69-FITC (11-0699, eBioscience, 1:40), CD161-APC (130-098-908, Miltenyi Biotech, 1:100), CD161-PE (130-099-193, Miltenyi Biotech, 1:100), IFN-γ-FITC (130-091-641, Miltenyi Biotech, 1:50), IFN-γ-APCCy7 (502529 Biolegend, 1:50), Vα7.2-PE/PeCy7/APC/FITC (351705/351711/351707/351703, Biolegend, 1:50). Granzyme B-APC (MHGB05, Invitrogen), IL-18Ra-APC (17-7183-41, eBioscience, 1:50), TNF-α-PeCy7 (502929, Biolegend, 1:100). All data was acquired on a MACSQuant (Miltenyi Biotech) or a BD FACSVerse (BD) and analyzed on FlowJo (Tree Star Inc.). Gating strategy is shown in Supplementary Fig. 12 .
10
Immunophenotyping of Mast Cells
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