Red blood cell (RBC)-lysed blood and spleen samples were stained for 30 min at 4°C with mAb against the following molecules: CD44 (clone IM7, BD), CD127 (clone A7R34, Biolegend), KLRG1 (clone 2F1, BD), and CD8α (clone 53-6.7, eBioscience). H-2Db LT359 tetramers were provided by the NIH Tetramer Core Facility (Atlanta, GA). Samples were acquired on a LSRII instrument (BD Biosciences) and data analyzed using FlowJo software (TreeStar, Inc., Ashland, OR). For intracellular cytokine staining (ICS), cells were stimulated for 5 h with LT359-368 peptide (1 µM) in the presence of Golgiplug (BD Biosciences). Cells were surface stained, permeabilized and fixed using the Cytofix/Cytoperm kit (BD Biosciences), and stained with mAbs specific for IFN-γ (clone XMG1.2, eBioscience) and TNF-α (clone TN3-19.12, BD Biosciences). Nucleated cells from blood and spleen samples were counted with TC20™ automated cell counter (Bio-Rad). The number of LT359 tetramer+ CD8 T cells was calculated as the total number of nucleated cells × the percentage of specific subpopulation out of total population as determined by using FlowJo software.
Tnf α clone tn3 19
Manufactured by BD
Sourced in Japan
TNF-α (clone TN3-19.12) is a monoclonal antibody that specifically binds to the tumor necrosis factor alpha (TNF-α) cytokine. TNF-α is a pro-inflammatory cytokine involved in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Ifn γ clone xmg1, by Thermo Fisher Scientific (2 mentions)
Cd8α clone 53 6, by Thermo Fisher Scientific (1 mentions)
Cd44 clone im7, by BD (1 mentions)
Cd127 clone a7r34, by BioLegend (1 mentions)
Klrg1 clone 2f1, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Golgiplug, by BD (1 mentions)
Tc20 automated cell counter, by Bio-Rad (1 mentions)
Lsrii instrument, by BD (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Agilent Technologies (1 mentions)
Hrp conjugated avidin, by Vector Laboratories (1 mentions)
Tnfα clone mp6 xt22, by BD (1 mentions)
3 protocols using tnf α clone tn3 19
1
Phenotypic Characterization of CD8+ T Cells
2
Serum Antibody and Cytokine Measurement
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Serum levels of anti-SSA/Ro and anti-SSB/La antibodies (Alpha Diagnostics International, San Antonio, TX, USA) were determined using ELISA kits according to the manufacturer’s protocol. Serum inflammatory cytokines in mice sera were measured by sandwich-ELISA using capture antibodies against IL-1β (clone B122; eBioscience, Affymetrix Japan, Tokyo, Japan), IL-5 (clone TRFK5; eBioscience), IL-10 (clone JESS-16E3; eBioscience), IL-17A (clone 17CK15A5; eBioscience), IFN-γ (clone XMG1.2; eBioscience), TNF-α (clone TN3-19.12; BD Pharmingen), and biotinylated-detection antibodies against IL-1β (clone 13-7112-85; eBioscience), IL-5 (clone TRFK4; eBioscience), IL-10 (clone JESS-2A5; eBioscience), IL-17A (clone 17B7; eBoiscience), IFN-γ (clone R4-6A2; eBioscience), TNF-α (clone MP6-XT22; BD Pharmingen). All antibodies were diluted 1:1000 in PBS containing 10% fetal calf serum (Gibco, Thermo Fisher Scientific, Tokyo, Japan). Biotinated antibodies were detected by HRP-conjugated avidin (Vector laboratories, Burlingame, CA, USA) and visualized by 3,3′,5,5′-tetramethylbenzidine (Dako), where the reaction was stopped with 2 M H2O2. Microtitier plate reader (Vmax, Molecular Devices, Tokyo, Japan) at 450 nm was used to measure the optical densities.
3
Tumor Immune Cell Profiling by Flow Cytometry
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Tumors were digested to prepare cell suspensions with collagenase and hyaluronidase solution. Then, the cell suspensions were filtered through a cell mesh (70 µm, BD Biosciences). The antibodies to CD45 (clone OX-1), CD3 (clone G4.18), CD4 (clone OX-35), CD8 (clone OX-8), NKR-P1A (clone 10/78), FoxP3 (clone FJK-16s), IFN-γ (clone DB-1), and TNF-α (clone TN3-19.12) were obtained from BD Biosciences. For intracellular cytokine staining, the harvested cells were stimulated with PMA (50 ng/mL) and ionomycin (500 ng/mL) for 4 hours and incubated for 1 hour with brefeldin A (10 µg/mL). Then, the cells were permeabilized using an Foxp3 Fixation and Permeabilization Kit (BD Bioscience). Subsequently, Foxp3, IFN-γ, and TNF-α were stained. Flow cytometric analysis was performed using the FACS flow cytometer (LSRFortessa X-20, BD). The data were analyzed by FloJo Data Analysis Software (V.10; Ashland, Oregon, USA).
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