Naïve CD4+ T cells were isolated from 30 mL fresh peripheral blood obtained from healthy volunteers by magnetic cell sorting using a human naïve CD4+ T cell isolation kit II (Miltenyi Biotec, USA). The purity of naïve CD4+ T cells was greater than 95%, as determined by flow cytometry. Then, the isolated naïve CD4+ T cells were resuspended in RPMI 1640 medium containing 10% fetal bovine serum (Gibco, USA) and diluted to 5×105 cells/mL. The cells were cultured at 37°C in 48-well plates and stimulated with plate-bound anti-human CD3 mAb (OKT3; 5 µg/mL; eBioscience, USA) and anti-human CD28 mAb (5 µg/mL; eBioscience, USA) in the presence of IL-2 (5 ng/mL; PeproTech, USA) for four days. To assess the influence of ICOS signaling on CXCR3 expression, anti-ICOS mAb (ISA-3; 5 µg/mL; eBioscience, 16–9948-82, USA) was added to the culture medium.
Anti human cd3 mab okt3
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-human CD3 mAb (OKT3) is a monoclonal antibody that specifically binds to the CD3 complex on human T cells. It is a laboratory reagent used in flow cytometry and other immunological applications to identify and characterize T cells.
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Lab products found in correlation
Cd4 t cell isolation kit, by Miltenyi Biotec (2 mentions)
Soluble anti human cd28 cd28.2 mab, by BD (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Human na ve cd4 t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Isa 3, by Thermo Fisher Scientific (1 mentions)
Recombinant human il 2, by Thermo Fisher Scientific (1 mentions)
Cts optmizer t cell expansion sfm, by Thermo Fisher Scientific (1 mentions)
Retronectin, by Takara Bio (1 mentions)
Cts immune cell serum replacement, by Thermo Fisher Scientific (1 mentions)
Roferon a, by Bio-Techne (1 mentions)
Nk cell isolation kit, by Miltenyi Biotec (1 mentions)
Il 15, by Miltenyi Biotec (1 mentions)
4 protocols using anti human cd3 mab okt3
1
CXCR3 Expression in Activated Naive CD4+ T Cells
2
Generation and Infusion of iCART Cells
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PBMCs used to prepare primary CART cells were purchased from Precision for Medicine and the use of human samples was approved by ethical committee at Takeda Pharmaceutical Company Ltd. To transduce CAR, T cells were cultured on wells precoated with CD3/retronectin solution composed of 3.0 μg/ml anti-human CD3 MAb (OKT3; eBioscience) and 150 μg/ml retronectin (TAKARA), having T-cell activation medium composed of CTS OpTmizer T Cell Expansion SFM supplemented with CTS Immune Cell Serum Replacement (Thermo Fisher Scientific), supplemented with 400 IU/ml recombinant human IL-2 (PeproTech) for 3 days for activation. The activated T cells were transduced with retroviral vector harboring 19BBz, as described in the above section (Generation of iCART cells). The resulting cells were infused into NALM-6-bearing mice 4 days after transduction.
3
Isolation and Stimulation of CD4+ T-cells and NK-cells
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CD4+T-cells were isolated from frozen PBMCs. All CD4+T-cells were positively selected with a CD4+T-cell isolation kit (Miltenyi Biotec, Germany), yielding CD4+T-cell populations at a purity of 96–99%. Purified CD4+T-cells were stimulated as described previously (28 (link)) with 4 μg/mL plate-bound anti-human CD3 (OKT3) mAb (eBioscience, San Diego, CA) and 4 μg/mL soluble anti-human CD28 (CD28.2) mAb (Becton Dickinson) in presence of Recombinant human IL-2 (Proleukine, Chiron, Amsterdam, 100 U/mL) and recombinant human interferon alpha-2a (Roferon-A) and IFNλ2 (Biotechne, UK) at the indicated dose. After five days culture, CD38 and CD25 expression as well as the frequency of 7AAD+ cells were measured by flow cytometry on stimulated CD4+T-cells (Table S4). NK-cells were isolated from PBMCs. NK-cells were negatively selected with the NK-cell isolation kit (Miltenyi Biotec, Germany), yielding NK-cell populations at a purity of 96–99%. NK-cells were stimulated with IL-15 (Miltenyi Biotec, 10 ng/mL), IL-2 (Proleukine, Chiron, Amsterdam 100 U/mL) and recombinant human interferon alpha-2a (Roferon-A) at the indicated dose. After 3 days of culture, expression of CD56, CD95 and NKG2D was measured by flow cytometry (extended data Table 3a ).
4
CD4+ T Cell Activation and CCR5 Expression
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CD4+ T cells were isolated from frozen PBMCs. All CD4+ T cells were positively selected with a CD4+ T cell isolation kit (Miltenyi Biotec, Bergisch-Gladbach, Germany), yielding CD4+ T cell populations at a purity of 96–99%. Purified CD4+ T-cells were stimulated as described previously (20 (link)) with 4 μg/mL plate-bound anti-human CD3 (OKT3) mAb (eBioscience, San Diego, CA) and 4 μg/mL soluble anti-human CD28 (CD28.2) mAb (Becton Dickinson) in presence of Recombinant human IL-2 (Proleukine, Chiron, Amsterdam, 100 U/mL) and recombinant human interferon alpha-2a (Roferon-A 0.01–100 ng/mL). After four days culture, membrane CCR5 expression was measured by flow cytometry on CD4+ T cells.
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