Human Tfh cells were generated as previously described42 (link). Briefly, after overnight stimulation of naive CD4+ T cells with Dynabeads human T cell activator CD3/CD28 (Thermo Fisher) in complete RPMI medium, cells were transferred to 96-well plates coated with anti-CD3 and supplemented with 2 μg/ml of soluble anti-CD28 mAb (Biolegend), human recombinant IL-23 (25 ng/ml; Biolegend) and human recombinant TGFβ1 (5 ng/ml; Biolegend). After 4 d cells were collected and used for analysis.
Protocol detail
In vitro generation of human Tfh cells
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Variable analysis
independent variables
- Overnight stimulation of naive CD4+ T cells with Dynabeads human T cell activator CD3/CD28
- Cells transferred to 96-well plates coated with anti-CD3 and supplemented with 2 μg/ml of soluble anti-CD28 mAb
- Addition of human recombinant IL-23 (25 ng/ml)
- Addition of human recombinant TGFβ1 (5 ng/ml)
- Characteristics of the generated human Tfh cells
- Complete RPMI medium
- Positive control: Overnight stimulation of naive CD4+ T cells with Dynabeads human T cell activator CD3/CD28
- Negative control: Not specified
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