Anti tnfα bv421

Manufactured by BioLegend

Sourced in United States

Anti-TNFα-BV421 is a monoclonal antibody conjugated with the BV421 fluorochrome, designed for the detection of tumor necrosis factor alpha (TNFα) in various applications.

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TNFα-BV421

5 protocols using anti tnfα bv421

Multiparameter Flow Cytometry Profiling

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After cell culture, whole blood samples (10x10⁵ cells/mL) were incubated with the following panel: antibodies from BioLegend, San Diego, CA, USA: anti-CD45-PerCP (Clone: HI30), anti-CD3-AF647 (Clone: UCHT1), anti-CD14-PECy7 (Clone: M5E2), anti-CD4-APC/Cy7 (Clone: OKT4), anti-CD8-PE/Dazzle594 or BV510 (Clone: SK1). After 15 min in the dark, blood was washed once with PBS (1 mL) by centrifugation at 900×g for 5 min at room temperature (RT); then, Fixation buffer was added (100 μL, Cat: 420801, BioLegend, San Diego, CA, USA), and the samples were incubated for 20 min. Then, samples were washed twice with 1 mL of Intracellular Staining Perm Wash buffer (Cat: 421002, BioLegend, San Diego, CA, USA); after the second wash, they were mixed with monoclonal antibodies against cytokines from BioLegend, San Diego, CA, USA: anti-TNFα-BV421 (Clone: MAb11), anti-IL-6-PE (MQ2-13A5), anti-IL-1β-FITC (Clone:JK1B-1), anti-IFNγ-BV421 (Clone:4S. B3), anti-IL-8a-PE (Clone: E8N1). For the exclusion of dead cells, a Zombie Aqua fixable viability kit (BioLegend, San Diego, CA, USA) was added and incubated for 30 min at RT. Last, the mixture was washed once with PBS. At least 30,000 events were acquired in a FACSAria IIu (BD, San Jose CA) flow cytometer. Analysis was performed using Infinicyt^™ Software 1.8.

Cérbulo-Vázquez A., García-Espinosa M., Briones-Garduño J.C., Arriaga-Pizano L., Ferat-Osorio E., Zavala-Barrios B., Cabrera-Rivera G.L., Miranda-Cruz P., García de la Rosa M.T., Prieto-Chávez J.L., Rivero-Arredondo V., Madera-Sandoval R.L., Cruz-Cruz A., Salazar-Rios E., Salazar-Rios M.E., Serrano-Molina D., De Lira-Barraza R.C., Villanueva-Compean A.H., Esquivel-Pineda A., Ramirez-Montes de Oca R., Caldiño-Soto F., Ramírez-García L.A., Flores-Padilla G., Moreno-Álvarez O., Guerrero-Avendaño G.M, & López-Macías C. (2022). The percentage of CD39+ monocytes is higher in pregnant COVID-19+ patients than in nonpregnant COVID-19+ patients. PLoS ONE, 17(7), e0264566.

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Comprehensive Immune Cell Profiling

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All antibody staining was preceded by 15 min of 1:50 FcγR block in FC buffer, on ice. Extracellular antibodies were then added to FC buffer containing FcγR block, and incubated for 45 min on ice. Intracellular staining was accomplished after surface staining using the Foxp3 staining kit (eBioscience). Myeloid subsets were stained with anti-CD1lb-FITC, anti-Ly6C-AF700, anti-MR-PE-Cy7, anti-CD11c-PE/Dazzle594, anti-Ly6G-BV605 (BioLegend), anti-MHCII-eFluor450 (eBioscience), and anti-F4/80-APC (BioRad). To evaluate Tregs, cells were stained with anti-CD3-AF488, anti-CD4-APC, anti-CD25-PerCP-Cy5.5 (BioLegend), and anti-Foxp3-PE (BD Pharmingen). To assess T cell activation, total tumor cells were incubated for 4 hr at 37°C in a 5% CO2 incubator with Protein Transport Inhibitor Cocktail containing brefeldin A and monensin, or with Cell Stimulation Cocktail containing protein transport inhibitors and PMA/ionomycin (eBioscience). Cells were then stained with anti-CD3-AF488, anti-CD4-PE, and anti-CD8-PerCP-Cy5.5 followed by intracellular stain with anti-IFNγ-APC (BioLegend). In addition, 1E5 CD3⁺ cells were stimulated with 4E4 CD3/CD28 Dynabeads (ThermoFisher) for 3 days, followed by staining using anti-CD3-PerCP-Cy5.5, anti-CD8-BV650, anti-CD4-BV605, anti-IFNγ-APC, anti-TNFα-BV421 (BioLegend), and anti-GranzymeB-PE (eBioscience).

Barberi T., Martin A., Suresh R., Barakat D.J., Harris-Bookman S., Drake C.G., Lim M, & Friedman A.D. (2018). Absence of host NF-κΒ p50 induces murine glioblastoma tumor regression, increases survival, and decreases T cell induction of tumor-associated macrophage M2 polarization. Cancer immunology, immunotherapy : CII, 67(10), 1491-1503.

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Cytokine-Activated NK Cell Killing Assay

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Unstimulated (1 ng/mL IL-15) and stimulated (1 ng/mL IL-15 plus MICA/MICB and anti-NKp46 stimulation) NK cells were cocultured for 4 hours with K-562 tumor targets at 2:1 E/T ratio (500,000:250,000). Anti–CD107a-FITC antibody (BioLegend catalog 328606) was added at the start of incubation, followed by GolgiStop and GolgiPlug (BD Biosciences) after the first hour of incubation. Cells were washed with PBS, stained with Live/Dead fixable viability stain (Thermo Fisher Scientific), stained with surface antibodies, fixed, permeabilized, and stained intracellularly with anti–IFN-γ–BV650 (BioLegend catalog 502538) and anti–TNF-α–BV421 (BioLegend catalog 502932). An LSRII (BD) was used for flow cytometric analyses.

Myers J.A., Schirm D., Bendzick L., Hopps R., Selleck C., Hinderlie P., Felices M, & Miller J.S. (2022). Balanced engagement of activating and inhibitory receptors mitigates human NK cell exhaustion. JCI Insight, 7(15), e150079.

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Apoptosis and Tumor-Infiltrating Lymphocyte Analysis

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To assess apoptosis, the cells were cultured for 24 h in RIPM 1640 medium containing normal glucose (2 g/L) or low glucose (40 mg/L) levels. The cells were centrifuged and resuspended in 0.5 ml annexin V-binding buffer (KeyGEN Biotech, China). Thereafter, 5 μl annexin V-APC and 7-AAD were added to the samples and incubated at RT for 10 min in the dark. The samples were then analyzed on a FACS Caliber flow cytometer (BD Biosciences, US).
The lymphocytes from 4T-1-injected BALB/c mice were isolated as follows: the tumor tissues from the mice were sectioned and digested with 2 mg/ml collagenase IV and 100 ng/ml DNase I (sigma) at 37 °C for 30 min. The tissues were then added to RPMI 1640 media supplemented with 10% FBS and 0.5 mM EDTA and separated by discontinuous 30–70% Percoll (GE Healthcare). After stimulation with PMA/Ionomycin and BFA (sigma) for 6 h at 37 °C, the cells were harvested for surface staining and intracellular staining (BD Pharmingen), according to the manufacturer’s instructions. The antibodies were anti-CD45-BV510, anti-CD45-APCcy7, anti-CD3-APC, anti-CD3-BV650, anti-CD4-FITC, anti-CD4-PEcy7, anti-PD-1-BV785, anti-PD-1-PE, anti-TIGIT-BV421, anti-TCR α/β-PE anti-IFN-γ-BV785, anti-IFN-γ-APC, anti-TNF-α-BV421 and anti-TNF-α-APC (BioLegend). All the data were collected by BD FACS Celesta flow cytometry and processed by Flow Jo software.

Dong X., Li C., Deng C., Liu J., Li D., Zhou T., Yang X., Liu Y., Guo Q., Feng Y., Yu Y., Wang Z., Guo W., Zhang S., Cui H., Jiang C., Wang X., Song X., Sun X, & Cao L. (2023). Regulated secretion of mutant p53 negatively affects T lymphocytes in the tumor microenvironment. Oncogene, 43(2), 92-105.

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Comprehensive Immunophenotyping by Flow Cytometry

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The following monoclonal antibodies were used for flow cytometry analysis and cell sorting; anti-CD8a-Alexa Fluor 488 (300916, BioLegend, 1:200), anti-CD8a-FITC (FITC-65135, Proteintech, 1:50), anti-CD3-PE (317307, BioLegend, 1:200), anti-CD4-APC (300552, BioLegend, 1:200), anti-CD45RA-FITC (304148, BioLegend, 1:200), anti-CD45RO-PerCP/Cy5.5 (304222, BioLegend, 1:200), anti-PD-1-APC/Cy7 (329921, BioLegend, 1:50), anti-CD57-PerCP/Cy5.5 (359621, BioLegend, 1:200), anti-IFNγ-APC (502511, Biolegend, 1:200), anti-TNFα-BV421 (502931, BioLegend, 1:100), anti-CCR7-PE (353203, BioLegend, 1:50), anti-TIGIT-PerCP/Cy5.5 (372717, BioLegend, 1:50), anti-pan HLA (M0736, Dako, 1:200), control mouse IgG2a (401501, BioLegend, 1:1111), and anti-mouse IgG-FITC (406001, BioLegend, 1:200). LIVE/DEAD Fixable-Near IR Dead Cell Stain Kit (L10119, Thermo Fisher Scientific, 1:400), LIVE/DEAD Fixable-Aqua Dead Cell Stain Kit (L34957, Thermo Fisher Scientific, 1:400), and Human BD Fc Block (564220, BD Pharmingen, 1:50) was also used. MHC tetramer was prepared using QuickSwitch Quant HLA-A^*24:02 Tetramer Kit-PE (TB-7302-K1, MBL Life Science) and QuickSwitch Quant HLA-A^*24:02 Tetramer Kit-BV421 (TB-7302-K4, MBL Life Science) with appropriate peptides. CMV pp65_341-349 peptide (TS-0020-P, MBL) was purchased and all the other peptides were synthesized with a purity of >95% (Scrum Inc., or Toyobo).

Ogura H., Gohda J., Lu X., Yamamoto M., Takesue Y., Son A., Doi S., Matsushita K., Isobe F., Fukuda Y., Huang T.P., Ueno T., Mambo N., Murakami H., Kawaguchi Y., Inoue J.I., Shirai K., Yamasaki S., Hirata J.I, & Ishido S. (2022). Dysfunctional Sars-CoV-2-M protein-specific cytotoxic T lymphocytes in patients recovering from severe COVID-19. Nature Communications, 13, 7063.

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