Spleens, mesenteric lymph nodes, and iliac lymph nodes were isolated from C57Bl/6 mice after cervical dislocation. CD8+ T cells were isolated by using a negative selection magnetic CD8+ T-cell isolation kit (Milteny Biotec) according to the manufacturer’s protocol. About 0.3 × 106 cells were plated per well in a 96-well plate in a total volume of 200 μL complete RPMI (as stated above). In order to obtain undifferentiated Tc0 cells, the medium was supplemented with 20 U/mL IL-2 (Peprotech), 0.5 ng/mL IL-7 (Peprotech), 0.5 µg/mL soluble anti-CD3 (ThermoScientific), 0.5 µg/mL soluble anti-CD28 (ThermoScientific), and 10 µg/mL anti-IFN-γ (BioXcell). For Tc17 differentiation, the medium was supplemented with 20 U/mL IL-2 (Peprotech), 0.5 ng/mL IL-7 (Peprotech), 20 ng/mL IL-6 (Peprotech), 5 ng/mL TGF-β (BioLegend), 20 ng/mL IL-1β (Peprotech), 20 ng/mL IL-23 (R&D systems), 0.5 µg/mL soluble anti-CD3 (ThermoScientific), 0.5 µg/mL soluble anti-CD28 (ThermoScientific), 10 μg/mL anti-IL-4 (BioXcell), and 10 µg/mL anti-IFN-γ (BioXcell). The cells were incubated for 2 days at 37°C and 5% CO2, after which the medium was refreshed with the same cytokine stimulations, but without anti-CD3 and anti-CD28. The cells were incubated for one more day before analysis by flow cytometry or adoptive transfer.
Soluble anti cd28
Manufactured by Thermo Fisher Scientific
Sourced in United States, Australia
Soluble anti-CD28 is a laboratory reagent used in cell culture studies. It is an antibody that binds to the CD28 receptor on the surface of T cells, a key co-stimulatory molecule involved in T cell activation. The primary function of soluble anti-CD28 is to provide an exogenous co-stimulatory signal to T cells in vitro.
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Lab products found in correlation
Anti cd3, by Thermo Fisher Scientific (34 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (11 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (8 mentions)
Soluble anti cd3, by Thermo Fisher Scientific (6 mentions)
Soluble anti cd3e, by Thermo Fisher Scientific (6 mentions)
Tgf β, by R&D Systems (5 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (4 mentions)
Plate bound anti cd3, by Thermo Fisher Scientific (4 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (3 mentions)
Il 12, by Thermo Fisher Scientific (3 mentions)
Interleukin 6 (il 6), by R&D Systems (3 mentions)
Complete rpmi 1640 culture medium, by Thermo Fisher Scientific (3 mentions)
Anti il 4, by Thermo Fisher Scientific (3 mentions)
Facscalibur, by BD (3 mentions)
Golgistop, by BD (3 mentions)
Human tgf β1, by R&D Systems (3 mentions)
Mouse il 2, by R&D Systems (3 mentions)
Anti il 4 neutralizing antibody, by Thermo Fisher Scientific (3 mentions)
Anti ifnγ neutralizing antibody, by Thermo Fisher Scientific (3 mentions)
79 protocols using soluble anti cd28
1
Isolation and Differentiation of Murine CD8+ T Cells
2
Th1 and Th17 Cell Differentiation Assay
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Purified naïve CD4+ T cells were cultured under Th1 or Th17 cell differentiation condition. For Th1 cell differentiation, naïve CD4+ T cells were cultured with plate-bound anti-CD3, soluble anti-CD28 (5 µg/ml) and anti-IL-4 (10 μg/m.) antibodies (eBioscience) combined with recombinant IL-12 (10 ng/ml) (Peprotech) in 96 well plates (BD Pharmingen). For Th17 cell differentiation, naïve CD4+ T cells were cultured with plate-bound anti-CD3 (10 μg/ml), soluble anti-CD28 (5 μg/ml), anti-IFN-ϒ (10 μg/ml) and anti-IL-4 (10 μg/ml) antibodies (eBioscience) combined with recombinant TGF-β1(5 ng/ml) and IL-6 (10 ng/ml) (PeproTech) in 96 well plates (BD Pharmingen). The CD4+ T cells were cultured with or with LTP for 4 days and collected for RNA isolation.
3
Differentiation and Stimulation of CD4+ T Cells
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Naive CD4+ T cells (1 × 105 per well) were activated with anti-CD3 (Invitrogen, CA, USA) and soluble anti-CD28 (Invitrogen, CA, USA) for 72 h in 96-well microplates in the presence of polarization cytokines and blocking antibodies. Then the cells were restimulated by PMA plus ionomycin together with brefeldin A for 5 h, and intracellular cytokines were detected by flow cytometry. For Th1 differentiation, IL-12 (PeproTech, NJ, USA) and anti-IL-4 (BD Biosciences, CA, USA) were used; For Th2 differentiation, IL-4 (PeproTech, NJ, USA) and anti-IFN-γ (BD Bioscience, CA, USA) were applied; For Th17 differentiation, cells were stimulated with IL-6 (R&D Systems, Minneapolis, MN, USA), TGF-β (R&D Systems, Minneapolis, MN, USA) in the presence of anti-IL-4 and anti-IFN-γ antibodies. For DCA stimulation experiments, various dosages of DCA was added to the culture medium 24 h after CD4+ T cells activation and indicated inhibitors were added 30 min ahead of DCA treatment.
4
Phospho-Antibody Profiling of Activated T-Cells
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After pre-treatment with ACAT inhibitors for 7d as described above, PBMC were surface stained followed by a stimulation with 0.5 μg/ml plate-bound anti-CD3 (Invitrogen) and 5 μg/ml soluble anti-CD28 (invitrogen) for 30 min at 37°C. Cells were fixed and permeabilized with PhosphoFix/Perm Buffer (BD Bioscience) for 30 min at −20°C followed by intranuclear staining with saturated concentrations of phospho-antibodies in PBS.
5
OT-1 T Cell Adoptive Transfer Against EG7 Tumors
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EG7 cells are derived from EL4 cells and express chicken ovalbumin (OVA) as a tumor antigen. MC38 is a solid tumor predominantly composed of immunosuppressive cell types, such as monocytic myeloid-derived suppressor cells (M-MDSCs; ref. 21) . Congenic CD45.1 þ SJL (8-10-week-old) recipient mice were injected subcutaneously with 5 Â 10 5 EG7 cells. CD8 þ T cells were isolated from spleen and lymph nodes (dissociated as described above) of WT OT-1 or Peli1-KO/OT-1 mice using the Magnisort mouse CD8 þ T cell enrichment kit (Thermo Fisher Scientific). Isolated CD8 þ T cells were stimulated with platecoated anti-CD3 (5 mg/mL, eBioscience), soluble anti-CD28 (2 mg/mL, Invitrogen), and OVA257-264 peptide (1 mg/mL, InvivoGen) in RPMI medium supplemented with IL2 (20 ng/mL, R&D Systems) for 48 hours. 5 Â 10 6 WT OT-1 or Peli1-KO OT-1 cells were intravenously transferred into tumor-bearing mice 10 days after tumor cell injection. Tumors were monitored for 23 days postinoculation, and mice were sacrificed on day 23 to collect draining lymph nodes (DLN) and TILs. Tumor size was determined by caliper measurements 3 times a week.
6
Th2 Differentiation from Naïve T Cells
7
Differentiation of Th1 and Th17 Cells
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CD4+ T cells were purified from spleens of naïve DBA/1 mice by positive selection using microbeads against CD4 (Miltenyi Biotec) following the protocol provided by the manufacturer. Then, the purified naïve CD4+ T cells were seeded at 2 × 106 per well into 96-well U-bottom microplates with RPMI 1640 medium (Hyclone) supplemented with 10% fetal bovine serum (Hyclone, Carlsbad, CA), 100 units/ml penicillin, and 100 μg of streptomycin (all from Invitrogen-Gibco). Th1 differentiation was driven by the of naïve CD4+ T cells with 1 μg/ml plate-bound anti-CD3 (clone 2C11, eBioscience), 1 μg/ml soluble anti-CD28 (clone PV1.17, eBioscience), 10 ng/ml IL-12 (PeproTech), 5 ng/ml IL-2 (PeproTech), and 2 μg/ml anti-IL-4 antibody (clone 11B11, eBioscience). Th17 differentiation was driven by the stimulation of naïve CD4+ T cells with 1 μg/ml plate-bound anti-CD3, 1 μg/ml soluble anti-CD28, 50 ng/ml IL-6 (PeproTech), 10 ng/ml TGF-β1 (PeproTech), 2 μg/ml anti-IFN-γ antibody (clone R4-6A2, eBioscience) and 2 μg/ml anti-IL-4 antibody. DXM was used at the dose indicated at the beginning of the Induction. Cells were harvested on day 4 of DXM treatment and analyzed for intracellular cytokines while culture supernatants were examined for cytokine levels by ELISA assay.
8
Generation of Tc1/Trm-like Cells from Naive CD8 T Cells
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Naïve CD8 T cells were purified by magnetic selection from healthy donor peripheral blood mononuclear cells [PBMC] using the naïve CD8 T cell isolation kit [Miltenyi Biotec] and were >98% CD8+ and >98% CD45RA+. Naïve CD8 T cells were stimulated with plate-bound anti-CD3 [1 µg/ml], soluble anti-CD28 [1 µg/ml], and IL-2 [5 ng/ml, Peprotech]. Further additions of TGF-β [3 ng/ml, R&D Systems], IFN-β [10 ng/ml, R&D], all-trans retinoic acid [ATRA; 10 nM, Sigma], FICZ [AhR agonist; 100 nM, Tocris Bioscience] were made at the start of the 7-day culture. Cultured cells were washed in PBS, stained for viability and surface or intracellular markers as above. Tc1/Trm-like cells were analysed for cytokine production by re-stimulation with PMA [20 ng/ml] + ionomycin [400 ng/ml] + monensin [3 µM] for 4 h before staining using Foxp3 staining buffer set.
9
Isolation and Differentiation of Naïve CD4+ T Cells
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Whole blood leukopaks from healthy adult donors were purchased from the Pittsburgh Central Blood Bank, with the approval of the University of Pittsburgh Institutional Review Board. PBMCs were isolated from the buffy-coat by Ficoll-Paque Plus (GE Healthcare Bio-Science AB) density gradient centrifugation. CD4+ T cells were isolated with the naïve T Cell Isolation Kit (Miltenyi Biotec Inc., Bergisch Gladbach, Germany). The isolated naïve CD4+ T cells were cultured in 96-well plates at a density of 4 × 104 cells/well. For Th0 cell cultures, cells were activated with 10 μg/mL plate-bound human anti-CD3 (PeproTech, Cranbury, NJ) and 1 μg/mL soluble anti-CD28 (PeproTech, catalog 10311–20) for 5 days under nonpolarizing conditions. For Th1 or Th17 cell differentiation, cells were cultured utilizing Human Th1 or Th17 Cell Differentiation Kit (R&D Systems, Inc.) according to the manufacturer's instructions.
10
Th2 Differentiation from Naïve T Cells
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Enteric naïve T helper cells were purified and cultured in an anti-CD3 (2μg/mL; 17A2; Biolegend) pre-coated plate. For Th2 differentiation, cells were stimulated in the presence of soluble anti-CD28 (1μg/mL; 37.51), recombinant mouse IL-2 (10ng/mL) (Peprotech), IL-4 (50ng/mL), anti-IFN-γ (1μg/mL; R4-6A2) and anti-IL-12 (1μg/mL; C17.8) (Biolegend). Polarised Th2 cells were analysed after 4 days of differentiation.
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