Effect of B-cell Depleting Therapies on PLA2R-specific B Cells in Patients With Membranous Nephropathy

"For tetramer assembly, recombinant biotinylated PLA2R will be incubated with either streptavidin-PE (Agilent) or streptavidin-APC (Agilent) conjugates according to the NIH protocol (https://tetramer.yerkes.emory.edu/support/protocols#10).~PBMCs will be thawed and resuspended in antibody cocktail medium containing 7.5ug APC- and PE- PLA2R tetramers. Multicolor flow cytometry will be used to identify B-cell subsets with standard technique and equipment (FACS FortessaX20, BD Biosciences and FlowJo software). PLA2R-specific B cells will be determined as Tetramer double (PE/APC) positive B cells that bind with proportional fluorescent intensities to each tetramer. Each sample will be incubated with only streptavidin-PE/APC conjugates to exclude B cells binding to the fluorochrome portion of the tetramers. T cell- and NK/NKT cell phenotyping will be assessed by FACS using ad-hoc antibody panels."