Alexa750 conjugated anti cd19

Freshly isolated B cells were treated with Human Fc Receptor Binding Inhibitor to block the Fc receptor prior to staining. B cells were then stained with FITC-conjugated CD27, PE-conjugated anti-CCR10, PE-Cy7-conjugated anti-CD20, Percp-Cy5.5-conjugated anti-CD38, APC-conjugated anti-CXCR7, Alexa700–conjugated anti-CD3 (Biolegend, San Diego, CA, USA) and Alexa750–conjugated anti-CD19. Cell sorting was performed using FACS-Aria. Appropriate known positive and negative controls were used throughout. Sorted CD3⁻CD19⁺CD20⁻CD27^hiCD38^hiCXCR7⁺CCR10^low cells were suspended in complete medium containing alkaline phosphatase conjugated goat antibodies against human IgA, human IgG and human IgM (all from KPL, Gaithersburg, Maryland, USA) at 0.2 μg/ml, respectively, and then seeded in 96-well MultiScreen HTS plates (Millipore, Billerica, MA, USA) coated with 100μl of affinity purified goat anti-human IgA+IgG+IgM (H+L) (KPL) at a concentration of 4 μg/ml in PBS. For antigen-specific ASCs, wells were coated with 100μl of recombinant GST-PDC-E2 fusion protein. Plates were incubated for 16 h at 37 °C under 5% CO₂ prior to development with blue alkaline phosphatase substrate. The number of total IgG/IgA/IgM ASCs and PDC-E2 specific ASCs in each well was thence determined as noted earlier.

PBMC freshly isolated from whole blood were first incubated with anti-mouse CD16/32 to block the Fc receptor, then stained with fluorochrome-conjugated antibodies including FITC-conjugated IgD, PE-conjugated anti-CD27, APC-conjugated anti-CD3, and Alexa750–conjugated anti-CD19 (Biolegend, San Diego, CA, USA). Stained cells were analyzed using a FACScan flow cytometer. Data was acquired on a total of 10,000 events and analyzed utilizing CELLQUEST software. Known positive and negative controls were used throughout.