The CD4+CD25− effector T cells (Teff) and CD4+CD25+ (Treg) cells were isolated by fluorescence-activated cell sorting (FACS) from liver or MLN of SAMP and AKR mice. Teff cells were labeled with eFluor 670 (5-(6)-carboxyfluorescein diacetate succinimidyl ester analog, 0.5 μM) and cultured either with or without a 1:1 ratio of Tregs. αCD3− (1 μg/mL), and αCD28− (1 μg/mL) coated beads (Miltenyi Biotec) were added to cultures at a 1:0.8 Teff:bead ratio. Proliferation was determined after 3 days of culture by flow cytometric dye dilution assays.
α cd28
Manufactured by Miltenyi Biotec
Sourced in Germany
The α-CD28 is a monoclonal antibody that binds to the CD28 receptor on the surface of T cells. CD28 is a costimulatory molecule that plays a crucial role in T cell activation and proliferation. The α-CD28 can be used in various cell culture and immunological applications to stimulate and expand T cells.
Automatically generated - may contain errors
Lab products found in correlation
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
X vivo 10 media, by Lonza (1 mentions)
Negative selection, by Miltenyi Biotec (1 mentions)
Ficoll, by GE Healthcare (1 mentions)
Chir99021, by Bio-Techne (1 mentions)
S9129, by Merck Group (1 mentions)
Phosflow perm buffer 3, by BD (1 mentions)
Cytofix buffer, by BD (1 mentions)
8 protocols using α cd28
1
Evaluating Treg-mediated Suppression of Teff Cell Proliferation
2
Treg Suppression of Tconv Proliferation
3
Treg Suppression of Teff Proliferation
4
Multi-Cytokine ELISPOT Assay Protocol
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IFN-γ, IL-5, and IL-17 ELISPOT assays (Lophius Biosciences, Regensburg, Germany) were performed according to the manufacturer’s recommendations, with minor modifications as described before [16 ]. Briefly, 2 × 105 PBMCs were added to each well of the ELISPOT plate and stimulated with AfuLy (50 μg/mL) or proteinaceous antigens (30 μg/mL). Plates for IFN-γ or IL-5 ELISPOTs were supplemented with 1 μg/mL α-CD28 and 1 μg/mL α-CD49d co-stimulatory antibodies (both Miltenyi Biotec, Bergisch Gladbach, Germany), whereas no co-stimulatory antibodies were used for IL-17 ELISPOTs, following previously optimized protocols [17 (link)]. Phytohemagglutinin (PHA, 10 μg/mL, Sigma-Aldrich, St. Louis, MI, USA) served as a positive control. Unstimulated control wells contained PBMCs and co-stimulatory factors (if applicable), but no antigens. IFN-γ ELISPOTs were incubated for 24–26 h at 37 °C and 5 % CO2, whereas longer incubation (44–48 h) was used for IL-5 and IL-17 ELISPOTs. Staining was performed according to the manufacturer’s instructions and numbers of spot-forming cells (SFCs) per million PBMCs were determined with a Bioreader 5000a (BioSYS, Karben, Germany), using previously published readout settings [17 (link)].
5
Expansion of Activated Human T Cells
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All procedures involving human subjects were carried out in accordance with the Declaration of Helsinki. Human peripheral blood mononuclear cells from healthy donors were obtained, after acquiring informed consent, under a St. Jude Children’s Research Hospital protocol approved by the hospital’s institutional review board. Peripheral blood mononuclear cells were stimulated on CD3 (1 µg/mL, Miltenyi Biotec, Bergisch Gladbach, Germany) and αCD28 (1 µg/mL, Miltenyi Biotec) antibody-coated non-tissue culture treated 24-well plates (144530, Thermo Fisher Scientific, Waltham, WA, USA). Human interleukin (IL)-7 and IL-15 (10 ng/mL and 5 ng/mL, respectively) (Biological Research Branch, National Cancer Institute, Frederick, MD, USA) were added to cultures on day 2. On day 3, T cells were transduced with retroviral particles on plates coated with retronectin (T100A, Takara Clontech, Mountain View, CA, USA) in the presence of IL-7 and IL-15. On day 5 transduced T cells were harvested and were subsequently expanded with IL-7 and IL-15. Non-transduced T cells were activated with CD3/CD28 and expanded in parallel with IL-7 and IL-15. Cells were cultured for 7-10 days prior to being used for in vitro or in vivo experiments.
6
T Cell Proliferation Assay with Somatostatin
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Human peripheral blood mononuclear cells (PBMC) were separated by density centrifugation using Ficoll (GE health care). CD3+ T cells, CD4+ T cells, CD8+ T cells, naive T cell subsets and memory T cells, were all isolated using negative selection (Miltenyi biotec) with confirmed purities of > 97%. Cells were then labelled with Carboxyfluorescein succinimidyl ester (CFSE, ThermoFisher) to track cell proliferation. All cells were cultured in serum-free X-vivo 10 media (Lonza). T cells were plated in U bottom 96 well plate at 1x105 cells/well in a total volume 200ul of medium. Polyclonal activation beads (αCD2, αCD3 and αCD28, Bead to Cell Ratio: 1:2, Miltenyi) were used to stimulate T cells for 4 days. Reagents for cell culture include recombinant somatostatin (100nM, S9129, Sigma-Aldrich); SSTR3i (MK-4256, 2nM, AdooQ Bioscience); GSK3 inhibitor (CHIR 99021, 20nM, TOCRIS); IL-2 (100U/ml, ThermoFisher.).
7
Treg Suppression of Tconv Proliferation
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FACS-purified CD4+CD25− Tconv and CD4+CD25+ Treg cells were isolated from 10-week old mice. Tconv were labeled with eFluor 670 (CFSE analog. 0.5 µM) and cultured with or without a 1:1 ratio of Treg cells. α-CD3 (1 µg/ml) and α-CD28 (1 µg/ml)-coated beads (Miltenyi Biotec) were added to cultures at 1:4 bead:Tconv ratio. Proliferation was determined after 3 days of culture by flow cytometric dye dilution assay.
8
Assessing NKG2D Signaling in T Cells
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On day 10 of Th1 culture, NKG2D downstream signaling was assessed in WT and Klrk1−/− cells sorted as viable CD4+ CD94+ lymphocytes to reach ≥60% expression of NKG2D in WT cells. After sorting, cells were incubated with α-MULT-1 antibody (10 µg/ml; Thermo Fisher Scientific) and rested for 3 h at 37°C in FCS-free RPMI 1640 supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 50 µM β-mercaptoethanol. 105 cells/condition were stained with different combinations of the biotinylated α-CD3 (2–10 µg/ml, BioLegend), α-CD28 (10 µg/ml, Miltenyi Biotec), and α-CD314 (α-NKG2D, clone A10, 10 µg/ml, Thermo Fisher Scientific) antibodies, rested for 30 min at 37°C, and cross-linked by the addition of 20 µg/ml Streptavidin-UNLB (Southern Biotec). Cells were fixed with BD Cytofix buffer and permeabilized with BD Phosflow Perm III buffer (both BD Bioscience) and subsequently stained for pERK1/2 (PerCP-eFluor710 anti-mouse pERK1/2, clone MILAN8R, Thermo Fisher Scientific).
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