Anti cd19 alexa fluor 488

100 μL Raji cells or primary human B cells were seeded at 0.5 million cells/mL in a 96 well plate. Approximately 24 hours later, cells were harvested at 300 g for 3 minutes at 4°C, washed once with ice-cold PBS, stained with anti-CD19-Alexa Fluor 488 (Thermo Fisher) at 1:250 dilution in 20mM HEPES buffer (pH 7.4), 150 mM NaCl, and 0.1% BSA for 30 minutes on ice, washed once with PBS, and then analyzed on a Beckman Coulter CytoFLEX instrument.

HEK293T cells (RRID CVCL_0063) were seeded at 100,000 cells/well in 24 well plates 12–18 hours prior to transfection. Cells were transfected using Lipofectamine 2000 with either with either 1.5 μg of empty pcDNA3.1(+) vector, 0.75 μg of CD19 DNA and 0.75 μg of empty pcDNA3.1(+) vector DNA (CD19 condition), or 0.75 μg of CD19 DNA and 0.75 μg of CD81 DNA (CD19+CD81 condition). 36–48 hours after transfection, cells were harvested in phosphate buffered saline (PBS) supplemented with 3 mM EDTA, transferred to a 96 well V-bottom plate, and then washed twice with PBS. Cells were then incubated on ice for 20 minutes with 2 μg/mL anti-CD19-Alexa Fluor 488 (Thermo Fisher) and anti-CD81-APC (BioLegend, Catalog number 349510, RRID AB_2564021) in 20 mM HEPES buffer pH 7.4, containing 150 mM NaCl, and 0.1% BSA. Cells were washed two times with PBS and analyzed on a Beckman Coulter CytoFLEX.