Human peripheral blood mononuclear cells (PBMC) isolated from whole blood were incubated for 1hr with and without PRN694 and stimulated with plate-bound anti-CD3 (2.5 μg/mL) and soluble anti-CD28 (1.0 μg/mL) (BD Biosciences) for 18hr. Supernatants were collected, frozen, and analyzed using the Human InflammationMap® v.1.0 biomarker panel (Myriad RBM). For the IL-2 inhibition dose-response, PBMC were incubated for 1hr with a concentration range of PRN694 and stimulated either with anti-CD3/CD28 or 10 μM of thapsigargin (Sigma-Aldrich) for 18hr. Supernatants were analyzed for IL-2 using the AlphaLISA IL-2 kit (Perkin Elmer). For cytokine inhibition in vivo, triplicate CD-1 mice were administered a 20 mg/kg intraperitoneal dose of PRN694 followed either 1 or 6hr later by administration of 10 μg/mouse of anti-CD3 (R&D Systems). Plasma was collected 2hr after anti-CD3 injection and analyzed for cytokines using the RodentMap® v.3.0 biomarker panel (Myriad RBM).
Anti cd3 cd28
Manufactured by Merck Group
Sourced in United States
Anti-CD3/CD28 is a lab equipment product that serves as a cell activation and expansion tool. It is designed to stimulate T-cell proliferation and function in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd3, by BD (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Anti cd3, by R&D Systems (1 mentions)
Thapsigargin, by Merck Group (1 mentions)
Soluble anti cd28, by BD (1 mentions)
Brefeldine a, by Merck Group (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Fixation buffer, by Thermo Fisher Scientific (1 mentions)
Anti cd28, by Jackson ImmunoResearch (1 mentions)
Anti cd3, by Jackson ImmunoResearch (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Trichostatin a, by Merck Group (1 mentions)
Dynabeads human t cell activator cd3cd28, by Thermo Fisher Scientific (1 mentions)
Mouse il 2 ready set go kit, by Thermo Fisher Scientific (1 mentions)
Anti cd28, by Thermo Fisher Scientific (1 mentions)
Anti cd3, by Thermo Fisher Scientific (1 mentions)
Cd4 cd25 regulatory t cell isolation kit, by Miltenyi Biotec (1 mentions)
Pan t cell isolation kit, by Miltenyi Biotec (1 mentions)
Anti ifn γ, by BD (1 mentions)
Anti il 4, by BD (1 mentions)
6 protocols using anti cd3 cd28
1
Inhibition of T-cell Activation by PRN694
2
Cytokine Release Assay for PBMCs
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PBMCs were TCR-stimulated for 16 h with soluble 2 μg/mL anti-CD3 and 2 μg/mL anti-CD28 (both Sanquin), 10 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 μg/mL ionomycin (both Sigma-Aldrich), or left untreated. To measure intracellular cytokines, 10 μg/mL brefeldine A (Sigma-Aldrich) was added 1 h after addition of anti-CD3/CD28 or PMA/ionomycin. After incubation and cellular staining, cells were acquired on a Fortessa Cell Analyzer.
3
Spermidine Enhances T-cell Activation
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For intracellular staining, PBMCs were stimulated in R10 with anti-CD3 (1 μg/ml, Jackson Immuno Research) and anti-CD28 (1 μg/mL, Jackson Immuno Research) with or without 10 μM spermidine for 4 days. On day 4, cells were re-stimulated with the same concentrations of anti-CD3/CD28 for 6 hr at 37°C in the presence of 1 μg/mL brefeldin-A (Sigma-Aldrich). As a control, cells were left unstimulated. Following surface marker staining as described above, cells were fixed with 100 μL Fixation buffer (eBioscience) for 20 min at RT in the dark. Next, cells were permeabilized with 100 μL of 1x Permeabilization buffer (eBioscience) for 15 min in the dark at RT. Then, cells were resuspended in the intracellular antibody mix (anti-IFN-γ, anti-Granzyme B, anti-Perforin) and incubated for 30 min in the dark at RT. After being washed twice with Permeabilization buffer the cells were resuspended in 200 μL of FACS buffer for analysis.
4
Modulation of T cell IL-2 by IFNβ
5
Isolation and Differentiation of Murine T Cells
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Mouse splenocytes were isolated from 8–10-wk-old C57BL/6 mice. For VIM study, mouse splenocytes from 6–10-wk-old Vim−/−, and WT mice were isolated. Mice were maintained in the Central Animal Laboratory at Turku University. CD4+CD62L+ T cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) was used to obtain naïve T cells. The cells were activated with plate-bound anti-CD3 (1 μg/mL, BD PharMingen, San Diego, CA) and anti-CD28 (1 μg/mL) for all Th0, Th17, and iTreg conditions. For Th17 polarization, IL6 (30 ng/ml, PeproTech, UK), TGFβ1 (5 ng/ml), and neutralizing antibodies anti-IL4 and anti-IFNγ (both at 10 μg/mL, BD PharMingen, San Diego, CA) were added, and cells were cultured for 3 d. For iTreg conditions, IL2 (10 ng/ml, R&D system, Minneapolis, MN) and TGFβ1 (20 ng/ml) were used, and cells were cultured for 7 d, followed by restimulation with anti-CD3/CD28 and culturing with fresh cytokine-containing medium for another 3 d. All cell cultures were performed in IMDM supplemented with 10% fetal calf serum, 2 mM glutamine, 100 iu/mL penicillin, 0.1 mg/mL streptomycin (Sigma, St Louis, MO), and 2.5 μM β-mercaptoethanol.
6
Stimulation of Immune Cells with PGE2, TCR, and IL-2
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Peripheral blood (approximately 50 ml) was drawn from patients pre-operatively. Peripheral blood mononuclear cells (PBMCs) (24-100×10 6 cells) from patients and blood donors were isolated by Isopaque-Ficoll (Lymphoprep, Axis-Shield PoC, Oslo, Norway) gradient centrifugation. Plasma was isolated from the samples and frozen at -80 °C in order for PGE 2 levels to be measured at the same time for all study objects. Cells were re-suspended in RPMI 1640 medium (Life Technologies, Carlsbad, CA, USA) supplemented with 1% fetal bovine serum, 1 mM sodium pyruvate, 0.9% (v/v) MEM non-essential amino acids, 100 U/ml penicillin and 100 U/ml streptomycin for cell stimulation (all Life Technologies). Cells were subsequently left unstimulated or subjected to five different stimulation conditions at 37 °C (see Fig. 2A); (1) PGE 2 (10 µM, Sigma-Aldrich, St. Louis, MO, USA) for 10 min, (2) crosslinking of biotinylated anti-CD3 (1 µg/ml, clone OKT-3, custom affinity-purified and biotinylated by Diatec Monoclonals AS, Oslo, Norway) and biotinylated anti-CD28 (5 µg/ml, clone CD28.2, eBioscience, San Diego, CA, USA) with avidin (50 µg/ml, Zymed Laboratories, Life Technologies) for 1 min, (3) a combination of PGE 2 (10 µM, 10 min) and TCR co-stimulation (anti-CD3/CD28, 1 min), (4) IL-2 (100 IE, Sigma-Aldrich) for 30 min or (5) a combination of PGE 2 (10 µM, 40 min) and IL-2 (100 IE, 30 min).
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