B cells and pDCs were resuspended in RPMI 1640 medium supplemented with 10% FBS, 1x penicillin/streptomycin, 2mM L-glutamine, 5mM HEPES (all from Life Technologies) and 55 μM β-mercaptoethanol (Sigma) and transferred into U-bottom 96-well plates. 7.5 x 104 B cells and 3.75 x 103 pDCs were added to each well, along with 1.5 nM mega CD40L (Enzo Biosciences). ICs generated by combining 2.5 μg/mL of IgED with 1 μg/mL of CG50 plasmid DNA were added to the co-cultures. Cells were cultured for 7 days at 37°C and 5% CO2, washed and stained with CD123-FITC (clone 7G3), CD19-BV421 (clone HIB19), CD27-PerCPCy5.5 (clone M-T271), IgD-PE (clone IA6-2) and CD38-APC (clone HIT2), all from BD Biosciences. For flow cytometry assessment of B cell and plasma cell numbers, co-cultured cells were acquired for a fixed amount of time. B cells were defined as CD123− CD19+ and plasma cells were defined as CD123−CD19+ CD27hi CD38hi cells. Supernatants were recovered at Day 7 for indirect quantification of IgM by ELISA as described previously48 (link). Anti-human IFN-α receptor (IFNAR) antibody (MedImmune LLC)49 (link) anti-IL6 antibody (clone 6708, R&D Systems), or an anti-IL6 antibody (MedImmune LLC)50 (link) were used at final concentrations of 10 μg/mL.
Cd123 fitc clone 7g3
Manufactured by BD
CD123-FITC (clone 7G3) is a fluorochrome-conjugated monoclonal antibody that binds to the CD123 antigen. CD123 is the alpha subunit of the interleukin-3 receptor, which is expressed on various cell types, including hematopoietic progenitor cells and certain leukemia cells. The FITC (fluorescein isothiocyanate) fluorochrome is used to label the antibody, enabling its detection and quantification in flow cytometry applications.
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2 protocols using cd123 fitc clone 7g3
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In Vitro B Cell Differentiation Assay
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In Vitro B Cell Differentiation Assay
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B cells and pDCs were resuspended in RPMI 1640 medium supplemented with 10% FBS, 1x penicillin/streptomycin, 2mM L-glutamine, 5mM HEPES (all from Life Technologies) and 55 μM β-mercaptoethanol (Sigma) and transferred into U-bottom 96-well plates. 7.5 x 104 B cells and 3.75 x 103 pDCs were added to each well, along with 1.5 nM mega CD40L (Enzo Biosciences). ICs generated by combining 2.5 μg/mL of IgED with 1 μg/mL of CG50 plasmid DNA were added to the co-cultures. Cells were cultured for 7 days at 37°C and 5% CO2, washed and stained with CD123-FITC (clone 7G3), CD19-BV421 (clone HIB19), CD27-PerCPCy5.5 (clone M-T271), IgD-PE (clone IA6-2) and CD38-APC (clone HIT2), all from BD Biosciences. For flow cytometry assessment of B cell and plasma cell numbers, co-cultured cells were acquired for a fixed amount of time. B cells were defined as CD123− CD19+ and plasma cells were defined as CD123−CD19+ CD27hi CD38hi cells. Supernatants were recovered at Day 7 for indirect quantification of IgM by ELISA as described previously48 (link). Anti-human IFN-α receptor (IFNAR) antibody (MedImmune LLC)49 (link) anti-IL6 antibody (clone 6708, R&D Systems), or an anti-IL6 antibody (MedImmune LLC)50 (link) were used at final concentrations of 10 μg/mL.
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