Indirect co-culture of T and B cells by supernatant was performed as described previously (10 (link)). In brief, FACS isolated T cell populations were plated at 5×104 cells per well and activated with 5 μg/mL soluble anti-CD3ε (17A2; BioXCell) and 1 μg/mL soluble anti-CD28 (37.51; BioLegend) for 72 hours. Supernatant from activated T cells was plated with 5×104 sorted WT or IL-21R-KO B cells per well and with 1 μg/ml anti-CD40 (IC10) and 5 μg/ml F(ab’)2 goat anti-mouse IgMμ (Jackson ImmunoResearch Laboratories) for 3 days for assessment of AID expression or 6 days for assessment of B cell antibody production by ELISA as described (10 (link)). For IL-4 depleted co-culture, T cells were plated with stimuli and 2 μg/mL anti-IL-4 (11B11) for 72 hours.
Soluble anti cd28 37.51
Manufactured by BioLegend
Soluble anti-CD28 (37.51) is a laboratory reagent used in cell culture and immunology research. It is an antibody that binds to the CD28 receptor on T cells, a key co-stimulatory molecule involved in T cell activation. This reagent can be used to stimulate T cells in vitro.
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Lab products found in correlation
3 protocols using soluble anti cd28 37.51
1
Co-culture of Activated T and B Cells
2
Activation and Proliferation of Naive T Cells
3
Isolation and Activation of Mouse T Cells
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Primary T cells were isolated from spleen and lymph node cell suspensions as previously described (29 (link)) using a CD4 isolation kit (Stemcell technologies). For T cell activation studies, 1 × 105 cells were cultured and activated as previously described (30 (link)) using plate bound anti-CD3 (145-2C11, Biolegend) and soluble anti-CD28 (37.51, Biolegend) at concentrations of 2 and 1 μg/ml, respectively. Bone marrow derived dendritic cells were differentiated as previously described (28 (link)) Briefly, 10 ×106 bone marrow cells were cultured in complete T cell medium (RMPI 1640, 10% FBS, 100 U/ml Streptomycin, 50 μM βme, 2 mM glutamine, and 1 mM sodium pyruvate) for 7 days in the presence of 10 ng/ml mouse GM-CSF and 2 ng/ml IL-4 (Peprotech). On day 3 and 5 fresh medium with cytokines was added. For co-culture experiments 2 ×104 BMDCs were incubated with 1 μg/ml anti-CD3 antibody (145-2C11, Biolegend) and 1 ×105 naïve CD4 T cells.
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