Splenic B cells were isolated from 5 or 12 weeks old MRL/Lpr mice using B Cell Isolation Kit (Miltenyi Biotec). The purity of isolated B cells was over 97%. B cells were stained with CSFE before co-culturing with purified TCRβ+CD138+ or TCRβ+CD138- cells in the presence of anti-CD3/CD28 antibodies (BD Biosciences), phorbol 12-myristate 13-acetate (PMA)/ionomycin, or autoantigens [1 μg/ml of DNA or SM (Immunovision)]. DNA was isolated from MRL/Lpr splenocytes by hyperthemo treatment at 42°C for 4 h. After 3 to 4 days of incubation, cells were analyzed for CFSE dilution by flow cytometry. In other assays, after 10 days of culture, culture supernatants were analyzed for antibody production as well as IL-2 and IFNγ secretion by ELISA (R&D Systems). In CD4 blocking expriments, Ultra-LEAF™ purified CD4 antibody (clone GK1.5, Biolegend), or control rat IgG (Sigma-Aldrich) were added to T and B cell co-cultures. In some co-culture experiments, T and B cells were incubated either as mixed or separated with 0.4 μm pore sized polyester Corning Transwell® membrane insert (Sigma-Aldrich).
Ultra leaf purified cd4 antibody clone gk1
Manufactured by BioLegend
The Ultra-LEAF™ purified CD4 antibody (clone GK1.5) is a lab equipment product manufactured by BioLegend. It is a purified monoclonal antibody that specifically binds to the CD4 receptor on the surface of T helper cells.
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Lab products found in correlation
2 protocols using ultra leaf purified cd4 antibody clone gk1
1
Splenic B Cell Activation Assay
2
MRL/Lpr Splenic B Cell Activation
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Splenic B cells were isolated from 5 or 12 weeks old MRL/Lpr mice using B Cell Isolation Kit (Miltenyi Biotec) and stained with CSFE before co-culturing with purified TCRβ+CD138+ or TCRβ+CD138-cells in the presence of anti-CD3/CD28 antibodies (BD Biosciences), phorbol 12-myristate 13-acetate (PMA)/ionomycin or autoantigens (1 µg/ml of DNA or SM (Immunovision)). DNA was isolated from MRL/Lpr splenocytes by hyperthemo treatment at 42°C for 4 hours. After 3-4 days of incubation, cells were analyzed for CFSE dilution by flow cytometry. In other assays, after 10 days of culture, culture supernatants were analyzed for antibody production as well as IL-2 and IFNγ secretion by ELISA (R&D Systems).
In CD4 blocking expriments, Ultra-LEAF™ purified CD4 antibody (clone GK1.5, Biolegend) or control rat IgG (Sigma-Aldrich) were added to T and B cell co-cultures. In some co-culture experiments, T and B cells were incubated either as mixed or separated with 0.4 µm pore sized polyester Corning Transwell® membrane insert (Sigma-Aldrich).
In CD4 blocking expriments, Ultra-LEAF™ purified CD4 antibody (clone GK1.5, Biolegend) or control rat IgG (Sigma-Aldrich) were added to T and B cell co-cultures. In some co-culture experiments, T and B cells were incubated either as mixed or separated with 0.4 µm pore sized polyester Corning Transwell® membrane insert (Sigma-Aldrich).
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