Sorted human memory CD4 T cells were cultured in 48-well plates (5×10 5 cells per well) containing platebound anti-CD3 (5 µg/ml, BD Bioscience) and soluble anti-CD28 (1 µg/ml, BD Bioscience) supplemented with 20 ng/ml IL-1β (Peprotech). For quantitative real-time PCR and ELISA, cells or supernatants were collected on days 1 and 2. Murine splenic CD4 T cells were enriched by a mojoSort™ mouse CD4 T cell isolation kit (Biolegend), cultured with plate-bound anti-CD3 (2 µg/ml, BD Bioscience) and soluble anti-CD28 (1 µg/ml, BD Bioscience), and stimulated with 20 ng/ml IL-1β (Peprotech) for 30 min to study the phosphorylation of signaling molecules.
Soluble anti cd28
Manufactured by BD
Sourced in United States, Germany
Soluble anti-CD28 is a laboratory reagent used in immunological research. It is a monoclonal antibody that binds to the CD28 receptor on the surface of T cells, a key co-stimulatory molecule involved in the activation of T cells. This product is intended for research use only and its specific applications should be evaluated by the researcher.
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Lab products found in correlation
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Anti cd3ε, by BD (8 mentions)
Rpmi 1640, by Thermo Fisher Scientific (5 mentions)
Interleukin 6 (il 6), by R&D Systems (5 mentions)
Tgf β1, by R&D Systems (5 mentions)
Interleukin 2 (il 2), by R&D Systems (5 mentions)
Recombinant il 6, by R&D Systems (5 mentions)
Ionomycin, by Merck Group (4 mentions)
Golgistop, by BD (4 mentions)
Anti il 4, by BioLegend (4 mentions)
Anti cd3, by Thermo Fisher Scientific (4 mentions)
Anti interferon γ, by BD (4 mentions)
Il 1β, by Thermo Fisher Scientific (3 mentions)
Macs beads and columns, by Miltenyi Biotec (3 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Clone 145 2c11, by BD (3 mentions)
Recombinant human tgf β1, by Thermo Fisher Scientific (3 mentions)
Macs separation column, by Miltenyi Biotec (3 mentions)
89 protocols using soluble anti cd28
1
Cytokine-Induced CD4 T Cell Activation
2
CD4 T Cell Activation with Anti-CD3/CD28 and IL-1β
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Sorted human memory CD4 T cells were cultured in 48-well plates (5 × 105 cells per well) containing plate-bound anti-CD3 (5 μg/ml, BD Bioscience) and soluble anti-CD28 (1 μg/ml, BD Bioscience) supplemented with 20 ng/ml IL-1β (Peprotech). For quantitative real-time PCR and ELISA, cells or supernatants were collected on days 1 and 2. Murine splenic CD4 T cells were enriched by a mojoSort™ mouse CD4 T cell isolation kit (Biolegend), cultured with plate-bound anti-CD3 (2 μg/ml, BD Bioscience) and soluble anti-CD28 (1 μg/ml, BD Bioscience), and stimulated with 20 ng/ml IL-1β (Peprotech) for 30 min to study the phosphorylation of signaling molecules or 48 h for cytokine production by ELISA or flow cytometry.
3
Evaluating PKS3053 Effect on T-cell Activation
4
Allogeneic CD4+ T Cell Activation Assay
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Naive T cells were isolated by negative selection with CD4 microbeads (Miltenyi Biotec) and labeled with the fluorescent dye CellTrace violet (CTV) at 5 mM according to manufacturer protocol. For coculture experiments, freshly isolated pDCs resuspended at 1 × 105 cells/100 µl RPMI 1640 were cultured in 96-well U-bottom plates alone, with CpG alone, or with CXCL4. After 24 h, pDCs were washed, and CTV-labeled allogeneic CD4+ T cells were added at 2 × 105 cells/200 µl ml RPMI 1640 for an additional 5 d.
Alternatively, anti-CD3 antibodies (5 µg/ml) were coated on wells of a 96-well U-bottom plate, and 2 × 105 CTV-labeled CD4 T cells were added and cultured for 3 d in the presence of soluble anti-CD28.2 (1 µg/ml; BD Biosciences) and with pDC supernatant collected from 24 h of culture. T cell proliferation was analyzed.
Alternatively, anti-CD3 antibodies (5 µg/ml) were coated on wells of a 96-well U-bottom plate, and 2 × 105 CTV-labeled CD4 T cells were added and cultured for 3 d in the presence of soluble anti-CD28.2 (1 µg/ml; BD Biosciences) and with pDC supernatant collected from 24 h of culture. T cell proliferation was analyzed.
5
Differentiation of Naive CD4+ T Cells
6
Isolation and Polarization of Murine CD4+ T Cells
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Spleens were removed from C57BL/6 mice and minced. Splenic red blood cells were removed with ammonium-chloride-potassium lysis buffer (0.15M NH4Cl, 1mM KHCO3, 0.1mM Na2 EDTA, pH 7.2∼7.4). The cells were resuspended in complete media containing RPMI 1640 supplemented with 5% fetal bovine serum and 1% antibiotics (all from Gibco, Grand Island, NY, USA). CD4+ T cells were isolated using a CD4+ T cell isolation kit (Miltenyi Biotec, San Diego, CA, USA), according to the manufacturer's protocol. The purity of isolated CD4+ T cells was assessed as > 95%. Negatively selected non-CD4+ cells were regarded as antigen-presenting cells (APCs) and irradiated at 3,000 rad before coculture. Isolated CD4+ T cells were stimulated with plate-bound anti-CD3 (0.5 mg/mL) and soluble anti-CD28 (1 mg/mL) (both from BD Biosciences) in the presence or absence of CsA (30 ng/mL) and KRGE (3 μg/mL or 10 μg/mL) for 72 h. For Th17 cell-polarizing condition, isolated CD4+ T cells were stimulated with plate-bound anti-CD3 mAb (0.5 mg/mL), soluble anti-CD28 mAb (1 mg/mL), anti-interferon (IFN) γ (2 mg/mL), anti-IL-4 (2 mg/mL), anti-IL-2 (2 mg/mL), IL-6 (20 ng/mL) (all from R&D Systems, Minneapolis, MN, USA), and transforming growth factor-beta (2 ng/mL, PeproTech, London, UK) for 72 h [16] (link), [17] (link).
7
T Cell Differentiation and Cytokine Profiling
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Naïve T cells were activated with plate-bound anti-CD3 (10 µg/mL) plus soluble anti-CD28 (2 µg/mL, BD Bioscience, San Jose, CA, USA) for 2 days. For T cell differentiation, CD4+ T cells were differentiated into Th17 or iTreg cells as previously reported [27 (link),29 (link)]. Briefly, CD4+ T cells were stimulated by anti-CD3/CD28 for 4 days with anti-IFN-γ and anti-IL-4 (both 10 µg/mL) in the presence of TGF-β1 (5 ng/mL, for Treg), or TGF-β1 plus IL-6 (10 ng/mL, for Th17) (all obtained from R&D Systems, Minneapolis, MN, USA). Cells were restimulated with phorbol 12-myristate 13-acetate (PMA, 25 ng/mL) plus ionomycin (500 ng/mL; Sigma, St. Louis, MO, USA) for 5 h with GolgiStop (BD Bioscience, San Jose, CA, USA) in the last 2 h for intracellular cytokine staining; or without GolgiStop for cytokine assays in the culture supernatants by enzyme-linked immunosorbent assay (ELISA). Where indicated, fasudil (Selleck Chemicals, Houston, TX, USA) was added to the cultures.
8
Quantifying T-cell Cytokine Secretion
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Spleen cells were activated with plate-bound anti-CD3 (5 µg/ml) plus soluble anti-CD28 (2 µg/ml, BD Bioscience) and stimulated with SEA (50 µg/ml) for 48 h. Cytokines in the supernatants were assayed by ELISA. IL-4 was assayed with OptEIA kits (BD PharMingen). ELISA plates were developed with TMB substrate (BD PharMingen) and read by a microplate reader (model 570, Bio-Rad).
9
Isolation and Activation of CD4 T Cells
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CD4 cells were isolated from spleen and lymph nodes by negative selection as previously described (Diehl et al., 2002 (link)). For Stat3+/+ and Stat3−/− mice, CD4 cells were purified by cell sorting (FACS-Aria; Becton Dickinson). CD4 cells were activated with plate-bound anti-CD3 (2C11) (5 μg/ml) and soluble anti-CD28 (1 μg/ml) (BD Pharmingen, San Diego, CA) mAbs in the presence or absence of IL-6 (50 ng/mL) (Miltenyi Biotec, Auburn, CA). Pharmacological inhibitors were added to culture 42 hr after activation and supernatants were harvested 6 hr later. APCs were purified by depleting CD4 and CD8 T cells using positive selection (Miltenyi), and followed by irradiation treatment (2000 rad). APCs and OT-II CD4 cells were co-cultured at 4:1 ratio in the presence of 5 μM OVA323-339 peptide (Barnden et al., 1998 (link)) with or without IL-6 (50 ng /mL) (Miltenyi) or anti-IL-6 (2.5 μg /mL) (BD Pharmingen).
Pharmacological inhibitors used were CGP-37157 (Tocris Bioscience, Ellisville, MO) (10 μM), CCCP (2 μM), rotenone (2 μM), antimycin (2 μM), Ru360 (10 μM), FK506 (InvivoGen, San Diego, CA) (10 nM), Stattic (10 μM).
Pharmacological inhibitors used were CGP-37157 (Tocris Bioscience, Ellisville, MO) (10 μM), CCCP (2 μM), rotenone (2 μM), antimycin (2 μM), Ru360 (10 μM), FK506 (InvivoGen, San Diego, CA) (10 nM), Stattic (10 μM).
10
T Cell Activation Effects on Breast Cancer Cells
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