Naïve CD4+ T cells collected from pooled spleen and lymph nodes were cultured in round-bottom 96-well plates at 1×105 cells/well. Plates were pre-coated overnight with anti-CD3ε (BD, clone 2C11), and washed with PBS before adding media and cells. All cells were cultured in IMDM media supplemented with 10% FCS (Gibco), Pen/Strep and 2 μM freshly added β-mercaptoethanol. For Th17 differentiation, this media was supplemented to achieve the following final concentrations: 1 μg/ml soluble anti-CD28 (clone 37.51, BD Pharmingen), 10 μg/ml anti-IFNγ (clone XMG1.2, BioLegend) and anti-IL-4 (clone 11B11, BioLegend), 1 ng/ml hTGFβ (Miltenyi), and 20 ng/ml rmIL-6 (Miltenyi). For Th1 polarization, the media contained instead 1 μg/ml soluble anti-CD28 (clone 37.51, BD Pharmingen), 10 μg/ml anti-IL-4 (clone 11B11, BioLegend), 1 ng/ml rmIL-2 and 10 ng/ml rmIL-12 (Miltenyi Biotec). PGE2 was obtained from Cayman chemical and stored and diluted according to manufacturer’s instructions.
Soluble anti cd28 clone 37
Manufactured by BD
Sourced in Germany, United States
Soluble anti-CD28 (clone 37.51) is a laboratory reagent used in immunological studies. It is an antibody that binds to the CD28 receptor on T cells. The core function of this product is to provide a tool for researchers to investigate the role of CD28 signaling in immune cell activation and function.
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Lab products found in correlation
Anti cd3 clone 145 2c11, by BD (2 mentions)
Plate bound anti cd3 clone 145 2c11, by BD (2 mentions)
Macs cd8 t cell isolation, by Miltenyi Biotec (1 mentions)
Bioplex technology, by Bio-Rad (1 mentions)
Anti ifn γ clone xmg1, by BioLegend (1 mentions)
Anti il 4 clone 11b11, by BioLegend (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Htgfβ, by Miltenyi Biotec (1 mentions)
Rmil 2, by Miltenyi Biotec (1 mentions)
Rmil 6, by Miltenyi Biotec (1 mentions)
Tgf β1 plus il 6, by R&D Systems (1 mentions)
Anti cd3 cd28, by R&D Systems (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
Golgistop, by BD (1 mentions)
Efluor 670, by Thermo Fisher Scientific (1 mentions)
Cd4 t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Rmil 33, by BioLegend (1 mentions)
Facsaria 2 cell sorter, by BD (1 mentions)
6 protocols using soluble anti cd28 clone 37
1
In vitro T cell differentiation
2
T Cell Proliferation and Cytokine Assay
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CD4+ T cells and CD8+ T cells were negatively sorted from the spleens and lymph nodes with the MACS CD4+ T Cell Isolation (130–090-860) and MACS CD8+ T Cell Isolation (130–104-075) Kits (Miltenyi Biotec, Bergisch Gladbach, Germany).
For in vitro proliferation, 5 × 105 isolated CD4+ and/or CD8+ T cells in 200 μl proliferation medium (RPMI supplemented with 10% FCS, 2 mM L-glutamine and 50 units/ml penicillin/streptomycin) were added in duplicate to 96-well plates precoated with anti-CD3 antibody (clone 2C11, 5 μg/ml) and soluble anti-CD28 (clone 37.51, 1 μg/ml; BD Pharmingen) was added. For TCR-independent T cell stimulation, 10 ng/ml phorbol 12,13-dibutyrate (PDBu) and 125 ng/ml of the calcium ionophore ionomycin were added to the media. Cells were harvested on filters after a 48-h stimulation period, pulsed with H3-thymidine (1 mCi/well) in the final 16 h and the incorporation of H3-thymidine was measured with a Matrix 96 direct β counter system.
IL-2 and IFN-γ production in mouse T cells after antibody stimulation was determined by BioPlex technology (BioRad Laboratories) from the supernatant.
For in vitro proliferation, 5 × 105 isolated CD4+ and/or CD8+ T cells in 200 μl proliferation medium (RPMI supplemented with 10% FCS, 2 mM L-glutamine and 50 units/ml penicillin/streptomycin) were added in duplicate to 96-well plates precoated with anti-CD3 antibody (clone 2C11, 5 μg/ml) and soluble anti-CD28 (clone 37.51, 1 μg/ml; BD Pharmingen) was added. For TCR-independent T cell stimulation, 10 ng/ml phorbol 12,13-dibutyrate (PDBu) and 125 ng/ml of the calcium ionophore ionomycin were added to the media. Cells were harvested on filters after a 48-h stimulation period, pulsed with H3-thymidine (1 mCi/well) in the final 16 h and the incorporation of H3-thymidine was measured with a Matrix 96 direct β counter system.
IL-2 and IFN-γ production in mouse T cells after antibody stimulation was determined by BioPlex technology (BioRad Laboratories) from the supernatant.
3
Investigating T Cell Activation and Cytokine Production
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Purified T cells were cultured in a 24-well plate (1 × 106 cells/well) for 72 h with complete RPMI containing 10% FBS. T cells from the same animal were cultured with either 10 nM 1,25(OH)2D3 solution or 0.1% ethanol (vehicle control) and with or without stimulation with 5 μg/mL plate-bound anti-CD3 (clone 145-2C11; BD Biosciences, San Jose, CA, USA) and 2 μg/mL soluble anti-CD28 (clone 37.51; BD Biosciences) antibodies during the entire culture period. Cells were incubated at 37°C in 5% CO2. After 72 h, the supernatant was collected for enzyme-linked immunosorbent assay (ELISA) analysis, and T cells were harvested for western blot analysis. CD4+ T cells were used for flow cytometric analysis. The experimental design and cell culture process are depicted in Fig. 1 .
4
Naïve T Cell Activation and Differentiation
5
Th17 Cell Differentiation from Naive CD4+ T Cells
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Naïve CD4+ T cells were isolated from the spleens of C57BL/6 mice using the CD4+ T Cell Isolation Kit II (Miltenyi Biotec). CD4+ CD25- T cells were sorted using a FACSAria II cell sorter (BD Biosciences). Purified cells were cultured for 5 days with plate-bound anti-CD3 (clone 145–2C11) (1 μg/mL) and soluble anti-CD28 (clone 37.51) (1 μg/mL) (both BD Bioscience). To induce Th17 differentiation, culture medium was supplemented with recombinant TGFβ (2 ng/mL), IL-21 (100 ng/mL), IL-23 (20 ng/mL), IL-6 (50 ng/mL), IL-1β (20 ng/mL), and blocking antibodies for IL-4 (200 ng/mL), IFN-γ (200 ng/mL), and IL-2 (200 ng/mL), respectively. Where indicated, rmIL-33 (10 ng/mL) (BioLegend) was added to the culture. On day 5, cells were counted, stained and analyzed by flow cytometry as described below. To assess cell proliferation, sorted-purified CD4+ CD25- T cells were labeled with the cell proliferation dye eFluor 670 (eBioscience) following the manufacturer’s instructions prior to 5-day stimulation with or without IL-33 under Th17 cell-skewing conditions. Loss of eFluor 670 was assessed by flow cytometry as indicative of cell proliferation.
6
Mouse CD4+ T Cell Isolation and Activation
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As described previously,12 (link) mouse CD4+ T-cells were purified using a CD4+ T cell isolation kit (mouse) (Miltenyi Biotec, Germany). In brief, spleens and lymph nodes were isolated from C57BL/6 mice (6–8 weeks-old) and ground with a syringe plunger to release splenocytes and lymphocytes into a culture dish. The homogenized cell suspensions were filtered with a cell strainer (nylon mesh with 70 µM pores) to remove debris. The suspended cells were centrifuged and the red blood cells were lysed with ACK lysis buffer. The residual cells were then purified by CD4+ T cell isolation kits following the manufacturer’s instructions. The purity of the isolated T cells was assessed by flow cytometric analysis. Then, CD4+ T cells were activated with plate-bound anti-CD3 (clone 145–2C11, 10 μg/mL) plus soluble anti-CD28 (clone 37.51, 2 μg/mL) (BD Bioscience, San Jose, CA).
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