PBMCs were incubated in FACS buffer (PBS supplemented with 0.5% bovine serum albumin) for 20 min at 4°C with the following antibodies: anti-CD4 (L200, BD Biosciences), anti-CD8 (RPA-T8, BD Biosciences), anti-CD3 (OKT3, Invitrogen), anti-γδTCR (B1.1, Invitrogen), and anti-CD19 (HIB19, eBioscience). The cells were subsequently washed twice and were resuspended with FACS buffer. The flow cytometry data was acquired on BD LSR Fortessa and analyzed by FlowJo V10. Dead cells were excluded by using Fixable Viability Dye (Invitrogen).
Anti cd3 okt3
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The Anti-CD3 (OKT3) is a monoclonal antibody that binds to the CD3 complex on the surface of T cells. It is commonly used in research applications to activate and stimulate T cell function.
Automatically generated - may contain errors
Lab products found in correlation
Ficoll paque plus, by GE Healthcare (4 mentions)
Easysep human cd4 t cell enrichment kit, by STEMCELL (3 mentions)
Anti cd28 clone 28, by Thermo Fisher Scientific (3 mentions)
Dynabeads regulatory cd4 cd25 t cell kit, by STEMCELL (3 mentions)
Anti cd28 10f3, by Thermo Fisher Scientific (2 mentions)
Human cd8 t cell enrichment kit, by STEMCELL (2 mentions)
Celltrace violet, by Thermo Fisher Scientific (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (2 mentions)
Anti cd28, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Corning (2 mentions)
Polybrene, by Merck Group (2 mentions)
Retronectin, by Takara Bio (2 mentions)
Cell culture media, by Corning (2 mentions)
Kod polymerase master mix, by Merck Group (2 mentions)
Anti cd28 cd28.2 activating antibodies, by Thermo Fisher Scientific (2 mentions)
48 protocols using anti cd3 okt3
1
Multicolor Flow Cytometry for Immune Phenotyping
2
Expansion of Primary Human NK Cells
3
Isolation and Activation of CD8+ T Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors using Ficoll-Paque Plus (GE Healthcare). CD8+ T cells were isolated from PBMCs by magnetic bead purification using a human CD8+ T cell enrichment kit (STEMCELL Technologies). The purity (> 95%) was evaluated by flow cytometry using an eF450-labelled antibody against CD8 (48–0087–42, Thermo Fisher). CD8+ T cells were cultured in complete Roswell Park Memorial Institute (RPMI)-1640 medium. Then, CD8+ T cells (1 × 105) were activated by stimulation with plate-bound anti-CD3 (OKT3, Thermo Fisher) at 2.5 µg/mL and anti-CD28 (10F3, Thermo Fisher) at 2 µg/mL in vitro.
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Stimulation and Activation of PBMCs
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PBMCs were thawed at 37°C in pre-warmed cell buffer, washed two times and stained using 5 μM CellTrace violet (ThermoFisher) per 2 × 106 cells in PBS (-Ca2+/−Mg2+, Gibco) for 5 min at 37°C. After, the cells were treated with human FcR blocking reagent (Miltenyi Biotec) for 10 min at RT, washed, counted and diluted to a final concentration of 106 cells/mL. 2 × 106 cells were incubated for 1-, 6-, 16- or 24 h in cell buffer alone, or with added 1 μg/mL lipopolysaccharide (Invivogen), 50 ng/mL Phorbol-myristate-acetate and 1 μg/mL Ionomycin (both Sigma-Aldrich), 100 μg/mL zymosan (Sigma-Aldrich), 10 μg/mL phytohemagglutinin-L (ThermoFisher), or 5 μg/mL anti-CD3 (OKT3, ThermoFisher) and anti-CD28 (CD28.2, ThermoFisher). Incubations were performed in ultra-low attachment 6-well plates (Corning), and wells were thoroughly scraped after incubating to detach PBMCs.
5
Apoptosis and Necrosis Induction Assay
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Cells were treated with 1 μM staurosporine for 4 hours (primary cells) or overnight (lymphoma cells) to induce apoptosis. Necrosis was induced by heat-killing at 60°C, for 30 minutes. Cell death was confirmed by flow cytometry detection of labeling with 7-Aminoactinomycin D (7-AAD) and Annexin V (Biolegend). Primary T cell activation was performed with 0.5 μg/ml anti-CD3 (OKT3, Thermo Fisher, 16-0037-85) + 0.5 μg/ml anti-CD28 (Thermo Fisher, 16-0288-85) in the presence of 500 IU IL-2 (Peprotech, UK). The following inhibitors were used at the concentrations indicated in the figures: blebbistatin (SigmaAldrich), H1152 (Tocris), Y27632 (Calbiochem), wortmannin (Calbiochem), latrunculin A (SigmaAldrich), cytochalasin D (Thermo Fisher Scientific).
6
Isolation and Activation of CD4+ T Cells
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CD4+ T cells were isolated by magnetic cell separation using CD4 Microbeads, human (Miltenyi Biotec, Bergisch Gladbach-Germany; 130-045-101). PBMCs or CD4+ T cells from healthy donors were cultured in RPMI 1640 medium (ThermoFisher Scientific (Gibco), Karlsruhe, Germany; 21875-034) supplemented with 10% heat-inactivated FBS (ThermoFisher Scientific (Gibco), Germany; 10500-064) and 1% penicillin/streptomycin (ThermoFisher Scientific (Gibco), Germany; 15140-122) and incubated with 10% allogenic SF or serum from healthy controls (HC) for 18 h. For proliferation assays, the CD4+ T cells were labeled with cell proliferation dye eFluor670 (5 µM) (ThermoFisher Scientific, Germany; 65-0840-85) according to the manufacturer’s instructions. The CD4+ T cells were stimulated with 5 µg/mL anti-CD3(OKT3) and 1 µg/mL anti-CD28(CD28.2) (ThermoFisher Scientific, Germany; 16-0037-85 and 16-0289-85, respectively); the cells were treated with 20 µM 4-OI or (DMSO) as a vehicle control for 72 h.
7
Isolation and Activation of CD8+ T Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from HLA-A24 healthy donors using Ficoll-Paque Plus (GE Healthcare, Chicago, IL, USA). CD8+ T cells were isolated from the PBMCs by magnetic bead purification using a Human CD8+ T Cell Enrichment Kit (STEMCELL Technologies, Vancouver, BC, Canada). HLA-A24 phenotype and purity (>90%) were checked by flow cytometry using anti-HLA-A24 (human) monoclonal antibody (mAb)-phycoerythrin (PE) (K0208-5; MBL International) and eF450-labeled antibodies against CD8 (48-0087-42; Thermo Fisher Scientific, Waltham, MA, USA). CD8+ T cells were grown in complete RPMI-1640 medium plus interleukin (IL)-2 (10 IU/mL) and activated (1 × 105 cells) by stimulation with plates coated with 2.5 μg/mL of anti-CD3 (OKT3; Thermo Fisher Scientific, Waltham, MA, USA) and 2 μg/mL of anti-CD28 (10F3; Thermo Fisher Scientific, Waltham, MA, USA) in vitro.
8
Measuring IL-2 Secretion in T Cell Activation
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CHO:huPD-L1 or CHO:mock cells were treated with 10 μg/mL of mitomycin C (M4287; Sigma-Aldrich) for 30 minutes to inhibit proliferation. After washing with PBS, cells were seeded into culture plates precoated with 0.25 μg/mL anti-CD3 (OKT3; 16–0037; eBioscience). The next day, 1.25 × 106/mL T cells and test constructs were added in assay medium (RPMI 1640 + 10% FBS + 1% Pen/Strep). After 48 hours of coculture, supernatants were collected and analyzed for IL2 secretion in an electrochemiluminescence (ECL) immunoassay (Mesoscale Discovery) using an IL2 DuoSet kit (DY202; R&D Systems). All incubation steps were performed at 37°C, 5% CO2.
9
Electroporation of Activated PBMCs
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PBMCs were collected under informed consent from healthy donors. PBMCs were stimulated with 600 U/ml IL-2 (rIL-2; R&D Systems, Minneapolis, MN) and 50 ng/ml anti-CD3 (OKT-3; eBioscience, San Diego, CA) in AIM-V medium (Life Technologies, Carlsbad, CA) supplemented with 2% human AB serum for 7 days. The concentration of rIL-2 was increased to 1,000 IU/ml on day 8.
Electroporation were performed with the Nucleofector device II (Lonza, Cologne, Germany). 10 × 106 PBMCs were activated for 8 days as described above and were thereafter re-suspended in 100 μL of Cell Line Nucleofector Solution V (Lonza, Cologne, Germany) and TCR mRNA was added at 200 μg/ml47 (link). The mixture was placed in a certified cuvette (Lonza, Cologne, Germany) and electroporated. After electroporation, cells were re-suspended in AIM-V medium (Life Technologies, Carlsbad, CA) supplemented with 2% human AB serum and 100 IU/ml rIL-2. Transfected cells were maintained in a humidified 37 °C and 5% CO2 incubator until flow cytometry analysis and/or co-culture experiments.
Electroporation were performed with the Nucleofector device II (Lonza, Cologne, Germany). 10 × 106 PBMCs were activated for 8 days as described above and were thereafter re-suspended in 100 μL of Cell Line Nucleofector Solution V (Lonza, Cologne, Germany) and TCR mRNA was added at 200 μg/ml47 (link). The mixture was placed in a certified cuvette (Lonza, Cologne, Germany) and electroporated. After electroporation, cells were re-suspended in AIM-V medium (Life Technologies, Carlsbad, CA) supplemented with 2% human AB serum and 100 IU/ml rIL-2. Transfected cells were maintained in a humidified 37 °C and 5% CO2 incubator until flow cytometry analysis and/or co-culture experiments.
10
Cytotoxic T-cell Degranulation Assay
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