Fresh peripheral blood from healthy volunteers was used to isolate peripheral blood mononuclear cells (PBMCs) by Ficoll-Hypaque density gradient centrifugation. A density of 1 × 106 PBMCs per well were seeded into 24-well culture plates with complete medium RPMI 1640 (containing 10%FBS, 100 U/mL penicillin and 100 μg/mL streptomycin) and stimulated with 100 ng/mL lipopolysaccharide (LPS)(Sigma, Missouri, USA) for 24 h to detect IL-1β, TNF-α and IL-6 production. Antigen stimulation was simulated using a cocktail of anti-CD3 and anti-CD28 antibodies (5:1) (Miltenyi Biotec, Palo Alto, CA) for 3 days at 37 °C in a humidified 5% CO2 incubator where after IL-10, IL-17 and IFN-γ were measured in the culture supernatants.
Anti cd3 and anti cd28 antibodies
Manufactured by Miltenyi Biotec
Anti-CD3 and anti-CD28 antibodies are cell culture reagents used in T cell activation and expansion. They bind to the CD3 and CD28 molecules on the surface of T cells, providing co-stimulatory signals that promote T cell proliferation and activation.
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4 protocols using anti cd3 and anti cd28 antibodies
1
Immune Cell Activation and Cytokine Profiling
2
Transduction of Activated T Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats obtained through the Gulf Coast Regional Blood Center, and they were cultured in 45% RPMI 1640 and 45% Click’s media (Invitrogen, Grand Island, NY), supplemented with 10% fetal bovine serum (FBS) and 100 U/ml IL-2 (Miltenyi Biotec). Buffy coats were tested negative for infectious viral pathogens. Retrovirus was produced by transient transfection of HEK293T cells using GeneJuice (EMD Millipore, Billerca, MA) with MoMLV gag-pol (PegPam3-e plasmid), RD114 envelope (RDF plasmid), and the pSFG retroviral vector encoding the transgenes. Supernatant was collected after 48–72 h to transduce T cells that were activated by stimulation with 0.5 μg/mL each of anti-CD3 and anti-CD28 antibodies (Miltenyi Biotec) in the presence of 100 U/mL IL-2. The spinfection technique was performed to transduce T cells with RetroNectin coating and expanded for 10–14 days post-transduction unless otherwise stated. For transductions with multiple vectors, the protocol was identical to the above, except the wells were coated with equal amounts of each retroviral supernatant.
3
Retroviral Transduction of PBMCs
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γ-Retroviral supernatants were produced
by transiently transfecting HEK-293T cells (3 × 106) with an RD114 envelope expression plasmid (a gift from M. Collins,
UCL), a Gag-pol expression plasmid (a gift from E. Vanin, Baylor College
of Medicine), and an SFG transgene plasmid. The transfection was carried
out using GeneJuice (Millipore) in accordance with manufacturer’s
guidelines.
Blood was obtained from buffy coats purchased from
NHSBT (NC07). PBMCs were isolated from buffy coats via density gradient
sedimentation by using Ficoll. PBMCs were activated using anti-CD3
and anti-CD28 antibodies (Miltenyi Biotec). 24 h post activation,
the culturing media was supplemented with 100 IU IL2 (2BScientific
Limited). At 72 h, 1 × 106 PBMCs were plated on retronectin-coated
6-well plates (Takara Clonetech) with retroviral vectors and centrifuged
at 1000g for 40 min. 72 h post-transduction, transduction
efficiency was assessed and PBMCs were maintained in complete RPMI
medium supplemented with 100 IU IL2.
by transiently transfecting HEK-293T cells (3 × 106) with an RD114 envelope expression plasmid (a gift from M. Collins,
UCL), a Gag-pol expression plasmid (a gift from E. Vanin, Baylor College
of Medicine), and an SFG transgene plasmid. The transfection was carried
out using GeneJuice (Millipore) in accordance with manufacturer’s
guidelines.
Blood was obtained from buffy coats purchased from
NHSBT (NC07). PBMCs were isolated from buffy coats via density gradient
sedimentation by using Ficoll. PBMCs were activated using anti-CD3
and anti-CD28 antibodies (Miltenyi Biotec). 24 h post activation,
the culturing media was supplemented with 100 IU IL2 (2BScientific
Limited). At 72 h, 1 × 106 PBMCs were plated on retronectin-coated
6-well plates (Takara Clonetech) with retroviral vectors and centrifuged
at 1000g for 40 min. 72 h post-transduction, transduction
efficiency was assessed and PBMCs were maintained in complete RPMI
medium supplemented with 100 IU IL2.
4
T-cell Activation Protocols
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Activation of T-cells was performed according to two methods. In one approach, polystyrene magnetic beads coated with an optimized mixture of monoclonal antibodies directed against ε chain of CD3 complex and anti-CD28 expressed on the T-cell surface (Dynabeads R Human T-Activator CD3/CD28, Thermo Fisher Scientific) were used to simulate antigen presentation and to activate the T-cells. Magnetic beads washed in PBS containing 5% human serum were mixed with cells at a ratio of 1:1 and incubated in PBS buffer containing 5% human serum at 4°C for 30 min, then for 4 min at 37°C. Cells were cyto-spun onto coverslips (ø12 mm) by centrifugation for 10 min at 800 g.
In the second method, coverslips (ø12 mm) were incubated with 0.01% poly-L-lysine for 24 h at room temperature (RT). At the indicated times, coverslips were washed and coated with anti-CD3 and anti-CD28 antibodies (Miltenyi Biotec) at a dilution of 1:500 by incubation for 1h at room temperature. After washing with PBS buffer, plates were used for seeding Jurkat T-cells (1.2x105).
In the second method, coverslips (ø12 mm) were incubated with 0.01% poly-L-lysine for 24 h at room temperature (RT). At the indicated times, coverslips were washed and coated with anti-CD3 and anti-CD28 antibodies (Miltenyi Biotec) at a dilution of 1:500 by incubation for 1h at room temperature. After washing with PBS buffer, plates were used for seeding Jurkat T-cells (1.2x105).
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