EasySep™ Human B Cell Enrichment Kit II Without CD43 Depletion

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	Following this B cells were enriched by magnetic microparticle purification using the EasySep Human B cell Enrichment Kit Without CD43 Depletion.

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Following this B cells were enriched by magnetic microparticle purification using the EasySep Human B cell Enrichment Kit Without CD43 Depletion.

Publication protocol

"Peripheral blood mononuclear cells (PBMC) were isolated using either sodium heparin tubes stored overnight followed by Ficoll gradient isolation (Cohort 1) or sodium heparin CPT Tubes isolated on the same day (Cohort 2). For in vitro stimulation of sorted
HCD DN2 cells, 500 mL of blood was spun at 2,500g for 5 minutes, the plasma layer was removed, and PBMC were isolated from the buffy coat by Ficoll gradient. Following this B cells were enriched by magnetic microparticle purification using the EasySep
Human B cell Enrichment Kit Without CD43 Depletion. Enriched B cells (HCD) or total PBMC were then sorted by first gating on CD19+ CD3- non-PC (not CD27++ CD38++) and then rNAV:IgD+ CD27- CXCR5+ CD11c-, DN1: IgD- CD27- CXCR5+ CD11c+ -,
DN2: IgD- CD27- CXCR5- CD11c++, aNAV: IgD+ CD27- CXCR5- CD11c++, and SWM: IgD- CD27+ CXCR5+ CD11c+ -. Antibodies and fluorochromes utilized are listed in Table S2 and the key resources table. Cells were stained at the manufacturers recommended concentration with 10% normal mouse serum to block Fc receptor and non-specific binding. Anti-mouse beads (Bangs) single stained with each fluorochrome were used for compensation. Intracellular staining was done using True-Nuclear Transcription Factor Buffer Set (Biolegend) for fixation and permeabilization. Ex vivo binding of 9G4+ antibodies to B cells was quantified by staining with
9G4 Pacific Blue (custom labeled) and comparing the median fluorescence intensity of resting naive and switched memory B cells.
Flow cytometry data was analyzed using the FlowJo software (Treestar)"

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