Purified CD19+ lymphocytes (1 × 105 cells/well) were cultured in 96-well plates in 50 μL medium (RPMI, 5%FBS) supplemented with 50 ng/mL rhIL-10 (Miltenyi Biotec), 20 U rhIL-2 (PeproTech), 0.1 μg/mL CD40L (Enzo Life Science) and 25 μg/mL anti-human IgM F(ab’)2 (Jackson ImmunoResearch). Purified CD4+ lymphocytes (2.5 × 105-3.5 × 105 cells/well) were cultured for 36 h with rhIL-2 (PeproTech, 50U/mL) at 37°C, 5% CO2 in 48-well plates that had been coated overnight with anti-CD3 (2 μg/mL) (BioLegend, clone OKT3) +/-anti-CD46 (2 μg/mL) (R&D Systems, clone 344519). To assess B-cell activation, 100 μL of T-cell supernatant was added to B cells and cultured for 5–10 days at 37°C, 5%CO2. To block the activity of IL-10, a neutralizing mAb to IL-10 (2.5 μg/mL) was used. To restore B-cell proliferation in mixtures containing T-cell supernatants rhIL-10 (1 μg/mL) was added.
Rhil 10
Manufactured by Miltenyi Biotec
RhIL-10 is a recombinant human interleukin-10 product. Interleukin-10 is an anti-inflammatory cytokine that regulates the immune response.
Automatically generated - may contain errors
2 protocols using rhil 10
1
Activated B-cell Proliferation Assay
2
B-cell Activation Assay Using T-cell Supernatant
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Purified CD19+ lymphocytes (1 × 105 cells/well) were cultured in 96‐well plates in 50 μL medium (RPMI, 5%FBS) supplemented with 50 ng/mL rhIL‐10 (Miltenyi Biotec), 20 U rhIL‐2 (PeproTech), 0.1 μg/mL CD40L (Enzo Life Science) and 25 μg/mL anti‐human IgM F(ab’)2 (Jackson ImmunoResearch). Purified CD4+ lymphocytes (2.5 × 105‐3.5 × 105 cells/well) were cultured for 36 h with rhIL‐2 (PeproTech, 50U/mL) at 37°C, 5% CO2 in 48‐well plates that had been coated overnight with anti‐CD3 (2 μg/mL) (BioLegend, clone OKT3) +/‐anti‐CD46 (2 μg/mL) (R&D Systems, clone 344519). To assess B‐cell activation, 100 μL of T‐cell supernatant was added to B cells and cultured for 5–10 days at 37°C, 5%CO2. To block the activity of IL‐10, a neutralizing mAb to IL‐10 (2.5 μg/mL) was used. To restore B‐cell proliferation in mixtures containing T‐cell supernatants rhIL‐10 (1 μg/mL) was added.
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