Spleens were excised from CAS- or SPI-offspring (at PND140) to determine T cell (Th1, Th2, Th17, and Treg) cytokine response. CD4+ T lymphocytes were purified from splenocytes by negative selection using anti-CD4 microbeads (Miltenyi, Auburn, CA). Purified CD4+ T cells (1 ×106/well) were activated with plate-bound anti-CD3 mAb (5 μg/mL, clone 145–2C11, BD Biosciences, San Diego, CA) in the presence of soluble anti-CD28 mAb (1 μg/mL, clone 37.51, BD Biosciences) for 48 h25 (link). Supernatants collected after T cell stimulation were stored at −70°C for cytokine analysis. Cells were then lysed, and RNA was prepared for Th1/Th2 gene expression analyses by quantitative RT-PCR (qRT-PCR).
Soluble anti cd28 mab
Manufactured by BD
Sourced in United States
Soluble anti-CD28 mAb is a laboratory reagent that binds to the CD28 receptor on T cells. It is used in research applications to study the role of CD28 signaling in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd3 mab, by BD (5 mentions)
Anti il 4 ab, by R&D Systems (2 mentions)
Anti ifn γ ab, by R&D Systems (2 mentions)
Recombinant il 6, by R&D Systems (2 mentions)
Anti cd4 microbeads, by Miltenyi Biotec (1 mentions)
Ficoll hypaque, by Cytiva (1 mentions)
Anti cd3 monoclonal antibody mab, by BD (1 mentions)
Ionomycin, by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by Miltenyi Biotec (1 mentions)
Texmacs medium, by Miltenyi Biotec (1 mentions)
Interleukin 2 (il 2), by BD (1 mentions)
B5887, by Merck Group (1 mentions)
S2889, by Merck Group (1 mentions)
P1880, by Merck Group (1 mentions)
Flat bottomed 96 well plate, by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Merck Group (1 mentions)
Gentamicin, by Merck Group (1 mentions)
Goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
24 well cell culture plate, by Corning (1 mentions)
8 protocols using soluble anti cd28 mab
1
Splenic T Cell Cytokine Response
2
Th17 Cell Differentiation Protocol
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Mouse spleens were collected for cell preparation and washed twice with phosphate-buffered saline. The spleens were minced and the red blood cells were lysed with 0.83% ammonium chloride. The total splenocyte fraction was filtered through a cell strainer and centrifuged at 1,300 rpm at 4 °C for 5 min. The cells were stimulated with 0.5 μg/ml plate-bound anti-CD3 monoclonal antibody (mAb) (BD Biosciences, San Jose, CA, USA), 1 μg/ml soluble anti-CD28 mAb (BD Biosciences), 2 μg/ml anti-IFN-γ Ab (R&D Systems, Minneapolis, MN, USA), 2 μg/ml anti-IL-4 Ab (R&D Systems), 2 ng/ml recombinant transforming growth factor β (TGF-β) (R&D Systems), and 20 ng/ml recombinant IL-6 (R&D Systems) for 3 days to establish Th17 polarization. Peripheral blood mononuclear cells were isolated from buffy coats using Ficoll-Hypaque® (Amersham Biosciences, Pittsburgh, PA, USA) and density gradient centrifugation.
3
Fetal Skin T Cell Activation Protocols
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Several stimulation protocols have been tested for fetal skin T cells (18–22 wk EGA). First, freshly isolated fetal skin cells were cultured in 96-well round-bottom plates (Merck) with TexMACS medium, IL-2 (2 ng/ml; both Miltenyi Biotec), and anti-CD3/CD28 mAb-coated microbeads (bead:cell ratio 1:1, Human T-Activator CD3/CD28, GIBCO BRL Dynabeads; Thermo Fisher Scientific). Second, freshly isolated fetal skin cells were cultured in 96-well flat-bottom plates (Beckton Dickinson Labware Europe) coated with an anti-CD3 mAb (10 µg/ml; BD Bioscience) and a soluble anti-CD28 mAb (3 µl/106 cells; BD Bioscience) in TexMACS medium and IL-2 for 3 d. Third, unsorted and flow-sorted fetal skin T cell subsets were cultured in the presence of IL-2 in 96-well flat-bottom plates in TexMACS medium with and without plate-bound anti-CD3 mAb (10 µg/ml; BD Bioscience) and a soluble anti-CD27 mAb (3 µl/106 cells; BD Bioscience) for 3 d. To bypass TCR/CD3 activation, unsorted fetal skin cells and flow-sorted T cell subsets were stimulated with PMA and ionomycin (1:500; Thermo Fisher Scientific) for 3 h. Irrespective of the stimulation protocol, cultured cells were analyzed for the expression of the activation marker CD69 by flow cytometry.
4
Isolation and Activation of Human CD8+ T Cells
5
Optimizing PBMC Proliferation Assays
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Total PBMC or LP cell populations (2 × 105 cell/well) were cultured for 3 or 2 days, respectively, at 37 °C and 5% CO2, in Iscove’s medium supplemented with 10% human FBS, 100 µg/mL gentamicin (Sigma-Aldrich) and 3 µg/mL L-glutamine (Sigma-Aldrich), without or with different concentrations of sodium acetate (S2889), sodium proprionate (P1880) or sodium butyrate (B5887, all from Sigma Aldrich). The cell cultures were stimulated polyclonally in flat-bottomed 96-well plates (Nunc, Roskilde, Denmark). Plates were precoated with 3 µg/mL goat anti-mouse IgG (Jackson Immunoresearch labs, West Grove, PA, USA), and 12.5 ng/mL mouse anti-CD3mAb (BD Pharmingen, San Diego, CA, USA) and 0.05 µg/mL soluble anti-CD28mAb (BD Pharmingen) were added together with the cells. T cell proliferation was measured by dilution of 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) dye. CFSE (Invitrogen) was added to 1 × 106 cells/mL PBMCs in a final concentration of 0.2 µM and incubated at 37 °C for 10 min prior to polyclonal stimulation. Cells were immediately washed in Iscove’s medium supplemented with 10% human FBS, 100 µg/mL gentamicin and 3 µg/mL L-glutamine after the CFSE incubation period. Proliferating cells were defined as cells that had undergone at least one cell division using non-stimulated cells as control.
6
Activation of T-cell Subpopulations
7
Th17 Polarization from UC PBMCs
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We treated 1 or 10 μM GSKʹ872 to 5×105 PBMCs from UC patients which were stimulated with anti-CD3 (0.5 μg/ml) or zVAD (20 μM) and TNF-α (10 ng/ml).
The cells were stimulated with 0.5 μg/ml plate-bound anti-CD3 mAb (BD Biosciences), 1 μg/ml soluble anti-CD28 mAb (BD Biosciences), 2 μg/ml anti-IFN-γ Ab, 2 μg/ml anti-IL-4 Ab, 2 μg/ml anti-IL-2 Ab (all from R&D Systems), 2 ng/ml recombinant TGF-β (R&D Systems), and 20 ng/ml recombinant IL-6 (R&D Systems) for 3 days to establish Th17 polarization.
The cells were stimulated with 0.5 μg/ml plate-bound anti-CD3 mAb (BD Biosciences), 1 μg/ml soluble anti-CD28 mAb (BD Biosciences), 2 μg/ml anti-IFN-γ Ab, 2 μg/ml anti-IL-4 Ab, 2 μg/ml anti-IL-2 Ab (all from R&D Systems), 2 ng/ml recombinant TGF-β (R&D Systems), and 20 ng/ml recombinant IL-6 (R&D Systems) for 3 days to establish Th17 polarization.
8
T Cell Proliferation Assay
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Splenocytes from WT and OX40-KO mice were cultured in triplicate in wells pre-coated with the anti-CD3 mAb (3 mg/mL, BD Biosciences) and the soluble anti-CD28 mAb (1 mg/mL, BD Biosciences) at 37 C with 5% CO 2 . After 72 hours of incubation and 12 hours before harvest, 5-ethynyl-2 0 -deoxyuridine (EdU) was added to the plates (final concentration of 50 mM). Cell proliferation was measured via EdU incorporation according to the manufacturer's instructions (EdU staining kit, RiboBio Corporation, Guangzhou, China).
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