Intracellular TNF-α in GD-OF was detected following treatment with Golgistop (Cat no. 554724; BD Biosciences, Franklin Lakes, NJ, USA) for 6 hours after bTSH stimulation. Cultures were then incubated for 12 hours, monolayers disrupted and centrifuged at 500g for 5 minutes, washed and resuspended in PBS containing 2% FBS with 0.1% sodium azide staining buffer. Cells were permeabilized and fixed with CytoFix/CytoPerm (Cat. no. 55472; BD Biosciences) for 20 minutes, and resuspended in 0.1 mL Perm/Wash buffer (Cat. no. 554723; BD Biosciences). Cells were incubated with anti-human TNF-α (Cat. no. 340511; BD Biosciences) Ab or isotype control Ab (Cat. no. 555748; BD Biosciences) for 1 hour and fixed with 1% paraformaldehyde. Flow cytometry of at least 106 events was conducted. Mean fluorescent intensity was calculated as a ratio of sample geometric mean fluorescence and isotype geometric mean fluorescence.
Anti human tnf α
Manufactured by BD
Sourced in United States
Anti-human TNF-α is a laboratory reagent used for the detection and quantification of human tumor necrosis factor-alpha (TNF-α) in biological samples. TNF-α is a pro-inflammatory cytokine that plays a crucial role in various physiological and pathological processes.
Automatically generated - may contain errors
Lab products found in correlation
Multiskan ascent microplate reader, by Thermo Fisher Scientific (2 mentions)
Cytofix cytoperm, by BD (1 mentions)
Golgistop, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
Recombinant human tnf α, by BD (1 mentions)
Elisa plate reader, by Bio-Rad (1 mentions)
Cd3 percp cd4 fitc cd8 pe trucount kit, by BD (1 mentions)
4 protocols using anti human tnf α
1
Intracellular TNF-α Detection in GD-OF
2
Measuring Serum TNF-α and Receptors in RMI
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The peripheral venous blood was collected at 8:00 AM and at 9:00 AM in the morning after an overnight fast (12 h) from all patients with RMI, and the serum samples were frozen and stored at -80°C until examination. According to manufacturer's specifications, plasma TNF-α levels were measured by ELISA using purified and biotinylated antihuman TNF-α monoclonal antibodies and recombinant human TNF-α (BD Biosciences Phar Mingen, San Diego, CA, USA). All samples were processed by ELISA plate reader (Bio-Rad, CA, USA) [28 (link)]. Serum was separated from blood samples and frozen at -80°C, and the levels of sTNFR-1 and sTNFR-2 were measured by ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's specifications. The concentration of TNF-α, sTNFR-1, and sTNFR-2 were expressed as ng/L [28 (link)].
3
Cytokine Release Quantification
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Supernatants from 24 h cultures were assayed for the release of IL1β, IL6, IL8, IL10, IL12p70 and GM-CSF using Bio-Plex human cytokine customized detection kits (Bio-Rad, Hercules, CA, US) following the manufacturer's protocol. In selected experiments, TNFα and IL10 levels were measured using an in-house ELISA with specific pairs of antibodies, including anti-human TNFα (Pharmingen, San Diego, CA) or anti-IL10 (Probiotek, Monterrey, MX), as previously described [18 (link)]. Supplemented RPMI-1640 was used as a negative control. Absorbance was read using a Multiskan Ascent Microplate Reader (Thermo Fisher Scientific, Waltham, MA) at 405 nm. The results are presented as the mean value of duplicate wells.
4
Comprehensive Phenotypic Characterization of CAR-T Cells
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After centrifugation, cells were suspended and washed three times with FACS wash buffer (1 x PBS containing 0.5% BSA and 0.03% sodium azide). Then, cells were stained with an antibody directed against MUC1 (BD, San Jose, CA, USA). To detect phosphorylated STAT3 and STAT5 in T cells, anti-human pSTAT3 (eBioscience, Santiago, CA, USA) and anti-human pSTAT5 (eBioscience, Santiago, CA, USA) monoclonal antibodies were used for staining. After 5 weeks of co-incubation with target cells, the ligands of PD-1, TIM-3 and LAG-3 on CAR-T cells were detected by anti-human CD279 (BD, CA, USA), anti-human CD366 (eBioscience, Santiago, CA, USA) and anti-human CD223 (eBioscience, CA, USA) antibodies, respectively. Moreover, anti-human TNF-α (BD, CA, USA) and anti-human IFN-γ (BD Bioscience, Franklin, NJ, USA) antibodies were used to investigate the level of intracellular TNF-α and IFN-γ in CAR-T cells. In addition, the CD3-PerCP/CD4-FITC/CD8-PE TruCOUNT kit (BD Bioscience, Franklin, NJ, USA) was used to determine the number of human CAR-T cells in peripheral mouse blood. CD4 + and CD8 + T cells were quantified according to the manufacturer’s instructions.
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