Pierce streptavidin-coated high-capacity flat-bottom 96-well plates (Thermo Fisher Scientific) were coated with 50 μl biotinylated MR1/5-OP-RU monomer (NIH Tetramer Core Facility) at 10 μg per ml in PBS (Sigma-Aldrich) overnight at 4 °C. Cryopreserved PBMCs were thawed in complete medium. CD8+ T cells were isolated using CD8 MicroBeads (Exp 3; Miltenyi Biotec) and CD3+ T cells using the REAlease CD3 MicroBead Kit (Exp 4 and validation experiments; Miltenyi Biotec) following the manufacturer’s instructions. Isolated CD8+/CD3+ T cells were washed in complete medium and resuspended at 1 × 107 cells per ml. One million (20 h stimulation) or 500,000 (68 h stimulation) cells were added per well to the appropriate 96-well plates (MR1/5-OP-RU-coated plate for TCR and TCR+cytokine stimulation, round-bottom plate for unstimulated and cytokine stimulation). IL-12 (50 ng ml−1; R&D Systems) and IL-18 (50 ng ml−1; R&D Systems) were added for cytokine stimulation; αCD28 (1 μg ml−1; clone: CD28.2; BioLegend) for TCR stimulation; IL-12, IL-18 and αCD28 for TCR+cytokine stimulation; and complete medium for unstimulated cells (final volume 200 μl per well). Cells were incubated for 20 h or 68 h at 37 °C, 5% CO2. For intracellular cytokine staining, brefeldin A (BioLegend) and monensin (BioLegend) were added for the final 4 h.
αcd28
Manufactured by BioLegend
Sourced in United States
αCD28 is a monoclonal antibody that binds to the CD28 receptor on T cells. CD28 is a co-stimulatory receptor that plays a crucial role in T cell activation and proliferation. The αCD28 antibody can be used in cell culture applications to stimulate T cell responses.
Automatically generated - may contain errors
Lab products found in correlation
Cyquant cell proliferation assay, by Thermo Fisher Scientific (2 mentions)
αcd3 145 2c11, by BioLegend (2 mentions)
Il 12, by Thermo Fisher Scientific (2 mentions)
Capture and detection antibodies, by BioLegend (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Akt kinase inhibitor, by Merck Group (2 mentions)
Rapamycin, by Santa Cruz Biotechnology (2 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (2 mentions)
C57bl 6, by Jackson ImmunoResearch (2 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
αcd28, by Cytek Biosciences (1 mentions)
Htgf β, by Thermo Fisher Scientific (1 mentions)
αil 4, by BioLegend (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
αifnγ, by BioLegend (1 mentions)
Il 1β, by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Pierce ldh cytotoxicity assay kit, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by BioConcept (1 mentions)
29 protocols using αcd28
1
Activation of MR1-restricted T cells
2
Cytokine Production by Activated CD4+ T Cells
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Human CD4+ T cells (3×105/well in 96-well plate) were activated with plate-coated αCD3 (5 μg/ml, BioLegend) and αCD28 (10 μg/ml, BioLegend) for 3 days in the presence of 10% fasted or refed serum from the subjects and 5% heat-inactivated FBS. Also, CD4+ T cells (2×105/well in 96-well plate) were differentiated into three T cell subtypes by incubation with the specific supplements for Th1 (20 ng/ml IL-12 and 10 μg/ml αIL-4), Th2 (10 ng/ml IL-4 and 10 μg/ml αIFNγ) or Th17 (20 ng/ml IL-6, 2 ng/ml TGF-β1, 10 ng/ml IL-1β, 10 ng/ml IL-23, 10 μg/ml αIL-4, and 10 μg/ml αIFNγ), respectively. They were differentiated for 3 days on plate-coated αCD3 and αCD28 in the presence of 10% fasted or refed serum from the subjects. All recombinant proteins and antibodies for differentiation media were purchased from Peprotech and eBioscience. Supernatants were collected, centrifuged to remove cells and debris, and stored at −80°C. The levels of cytokines, including IFNγ, IL-5, IL-13, IL-17, and IL-22 were measured by ELISA (R&D Systems). Results were normalized to cell number using the CyQuant cell proliferation assay (Invitrogen) or BCA protein assay (Pierce).
3
T Cell Lineage Specification Protocols
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For polarizations using plate-bound αCD3 + αCD28, tissue culture plates were coated with 5 µg/ml αCD3 (145-2C11; BioLegend) and 5 µg/ml αCD28 (37.51; Tonbo), incubated at 37°C for 4 h, and washed three times immediately before use. For differentiation to Th17 cells, naive CD4+CD62LhighCD44low T cells were incubated with IL-6 (20 ng/ml; Peprotech), hTGF-β (5 ng/ml; Peprotech), αIL-4 (10 µg/ml; 11B11; BioLegend), and αIFN-γ (10 µg/ml; XMG1.2; BioLegend), with or without IL-1β (2–10 ng/ml; Peprotech). For differentiation to Th1 cells, naive CD4+CD62LhighCD44low T cells were incubated with IL-12 (10 ng/ml; Peprotech), IL-2 (50 U/ml; eBioscience), and αIL-4 (10 µg/ml; 11B11; BioLegend), with or without IL-18 (0.3–3 ng/ml; Gibco). Th2 cell differentiation was achieved by incubating naive CD4+CD62LhighCD44low T cells with IL-4 (4 ng/ml; Peprotech), IL-2 (50 U/ml; eBioscience), and αIFN-γ (10 µg/ml; XMG1.2; BioLegend).
4
Single Cell Cytokine Production Assay
5
T-Cell Activation and Proliferation Assays
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For transfer of daughter cells, when confocal microscopy analysis of mitotic cells was performed or when cell proliferation was assessed, cells were cultured in T-cell medium (RPMI 1640 (Bioconcept), 2 mM L-Glutamine (Bioconcept), 2% penicillin-streptomycin (Sigma-Aldrich), 10% foetal bovine serum (Omnilab), 25 mM HEPES (Gibco, Life Technologies, Zug, Switzerland), 1× non-essential amino acids (Sigma-Aldrich), 50 µM β-Mercaptoethanol (Gibco), 1 mM sodium pyruvate (Gibco) supplemented with self-made human IL-2, and stimulated on plate-bound human Fc-ICAM-1 (50 µg/ml) (R&D Biosciences, Bio-Techne AG, Zug, Switzerland), α-CD3 (5 µg/ml) (145-2C11, BioLegend) and α-CD28 (5 µg/ml) (37.51, BioLegend) for 30–36 h. mTOR modulation was done by adding 20 nM of rapamycin (Santa Cruz, LabForce AG, Muttenz, Switzerland) or 1–2 µM of Akt-kinase inhibitor (Sigma-Aldrich) 12 h post stimulation. For adoptive transfer, 1 × 104 purified CD45.1 P14 cells (stimulated as indicated) were intravenously injected into naive C57BL/6 CD45.2 recipient mice. For cell proliferation assays, cells were stained with CellTrace VioletTM, CellTrace YellowTM or CellTrace VioletTM (Life Technologies) following manufacturer’s instructions prior to stimulation.
6
Characterizing CD8+ T cell Antitumor Function
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CD8+ T cells were sorted from tumor draining lymph nodes using a CD8 isolation kit (Stemcell, Vancouver, BC). 2 × 105 of CD8+ T cells were co-cultured with 2 × 104 IFN-ɣ treated MC38 tumor cells in 96-well PVDF plates (EMD Millipore, Billerica, MA) for 2 days. Spots were developed by following the manufacture’s protocol (BD Biosciences) and details have been described elsewhere5 (link).
For the suppression assay, MC38 tumors were collected 3 days post-IR and the CD11b+CCR2+Ly6Chi cell population was sorted using AriaIIIu 4-15 (BD Biosciences, San Jose, CA) at The University of Chicago Flow Cytometry Core facility. In the meantime, naïve CD8+ T cells derived from draining lymph nodes were labeled with CellTraceTM Violet dye (ThermoFisher) and washed. The sorted MDSCs and 2 × 105 labeled T cells were co-cultured in complete RPMI1640 with the presence of 1ug/ml αCD28 (Biolegend, clone 37.51) and 100 uM of β-mercapitoethanol (Sigma), in the wells of a flat-bottom 96-well plate coated with 5ug/ml αCD3 (Biolegend, clone 145-2C11). The amount of MDSCs was diluted by factor of 2 in serial dilutions, starting with 2 × 105 (1:1). Cells were harvested, stained for CD8+ T cells (Biolegend, clone 53-6.7) and analyzed by flow cytometry.
For the suppression assay, MC38 tumors were collected 3 days post-IR and the CD11b+CCR2+Ly6Chi cell population was sorted using AriaIIIu 4-15 (BD Biosciences, San Jose, CA) at The University of Chicago Flow Cytometry Core facility. In the meantime, naïve CD8+ T cells derived from draining lymph nodes were labeled with CellTraceTM Violet dye (ThermoFisher) and washed. The sorted MDSCs and 2 × 105 labeled T cells were co-cultured in complete RPMI1640 with the presence of 1ug/ml αCD28 (Biolegend, clone 37.51) and 100 uM of β-mercapitoethanol (Sigma), in the wells of a flat-bottom 96-well plate coated with 5ug/ml αCD3 (Biolegend, clone 145-2C11). The amount of MDSCs was diluted by factor of 2 in serial dilutions, starting with 2 × 105 (1:1). Cells were harvested, stained for CD8+ T cells (Biolegend, clone 53-6.7) and analyzed by flow cytometry.
7
CD4+ T Cell Isolation and Activation
8
Cytokine Production by Activated CD4+ T Cells
9
Isolation and Flow Cytometric Analysis of Immune Cells
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Spleens were aseptically removed from euthanised mice, mechanically homogenised and treated with ACK lysis buffer. Cells were then counted and seeded at 1.5 × 106 per well in complete RPMI, followed by treatment with 10 μg/mL brefeldin A (Sigma-Aldrich). Cells were stimulated with 5 μg/mL Ag85B/Acr (Lionex, Germany) or PPD (NIBSC, UK) with 2 μg/mL α-CD28 (Biolegend) for 6 hours before staining for flow cytometry. PMA/ionomycin treatment (200 ng/mL and 1 μg/mL, respectively – Sigma-Aldrich) was used as a positive control and for staining boundaries (data not shown).
For lymph node analysis, inguinal lymph nodes were excised from euthanised mice on the indicated day, followed by mechanical disruption, counting and immediate flow cytometric analysis.
For lymph node analysis, inguinal lymph nodes were excised from euthanised mice on the indicated day, followed by mechanical disruption, counting and immediate flow cytometric analysis.
10
Antibody and Protein Labeling Protocol
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Antibodies were desalted using Zebaspin Desalting Columns (7 kDa MWCO, Thermo Fisher Scientific, Cat. # 89882) and diluted to a concentration of 0.5 mg/ml, before incubation with a 40-fold molar excess of BG-GLA-NHS (New England Biolabs, Cat. # S9151S). To test different ratios of BG-GLA-NHS, 10-, 20-, 40-, or 80-fold molar excess of BG-GLA-NHS was added to the antibodies, The reaction was allowed to continue for 30 min at room temperature before it was stopped by removing the unreacted NHS-BG substrate using Zebaspin Desalting Columns (7 kDa MWCO). MegaCD40L (Enzo Lifesciences, Cat. # ALX-522-110-C010) was labeled with a 10-fold molar excess of NHS-GLA-BG, due to the high number of surface exposed lysines. αCD3 (BioLegend Cat. # 317302), αCD4 (BioLegend Cat. # 317402) and αCD19 (BioLegend Cat. # 302202) were each labeled with a 40-fold molar excess of NHS-GLA-BG. αCD28 (BioLegend Cat. # 302933) was labeled with a 20-fold molar excess of NHS-GLA-BG, due to significant precipitation at 40-fold molar excess. All BG-labeled antibodies were resuspended in PBS, and their concentration was determined using the IgG function on a Nanodrop 2000 device.
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