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 previously15 (link) with 4 µg/mL plate-bound anti-human CD3 (OKT3) monoclonal antibody (mAb; eBioscience, San Diego, CA, USA) and 4 µg/mL soluble anti-human CD28 (CD28.2) mAb (Becton Dickinson) in the 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 4 days of culture, membrane CCR5 and CXCR4 expression was measured by flow cytometry on CD4+T cells with an anti-CCR5 mAb (clone REA245 Miltenyi Biotec) and an anti-CXCR4 mAb (clone 12G5, BioLegend).
Soluble anti human cd28 cd28.2 mab
Manufactured by BD
Sourced in United States
Soluble anti-human CD28 (CD28.2) mAb is a monoclonal antibody that binds to the CD28 receptor on human cells. CD28 is a co-stimulatory molecule that plays a crucial role in T cell activation and proliferation. This antibody can be used in various immunological research and assay applications.
Automatically generated - may contain errors
4 protocols using soluble anti human cd28 cd28.2 mab
1
Isolation and Activation of CD4+ T Cells
2
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 ).
3
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.
4
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 with 4 µg/mL plate-bound anti-human CD3 (OKT3) monoclonal antibody (mAb; eBioscience, San Diego, CA, USA) and 4 µg/mL soluble anti-human CD28 (CD28.2) mAb (Becton Dickinson, Le Pont De Claix France) in the 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 dose19 (link). After 5 days of culture, CD38 and CD25 expression and the frequency of 7-AAD+ cells were measured by flow cytometry on stimulated CD4+T cells. Natural killer (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 (Supplementary Table 2a ).
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