Expansion of isolated B cells with the B Cell Expansion Kit, human

This application protocol describes the in vitro expansion of primary human B cells.  
Many downstream applications require expansion of B cells, due to their low frequencies in peripheral blood. In vitro cultivation of B cells can be time consuming and can often result in dead cells and low expansion rates. The B Cell Expansion Kit, human has a defined formulation that enables standardized expansion of isolated B cells. Expanded cells are fully functional and ready for downstream applications. The expansion rate reaches up to a 10-fold increase after 14 days of in vitro culture. The phenotype and characteristics of the expanded B cells are also comparable to the starting material, therefore preserving all B cell subsets. 

Protocol

Isolation of human PBMCs

For the isolation of human PBMCs refer to the protocol "Isolation of mononuclear cells from human peripheral blood by density gradient centrifugation".

Reagent preparation 

Preparation of PEB buffer (for staining of isolated B cells)

PEB buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS® BSA Stock Solution (# 130-091-376) 1:20 with autoMACS® Rinsing Solution (# 130-091-222). Keep buffer cold (2–8 °C). Always use freshly prepared buffer. 
Do not use autoMACS Running Buffer or MACSQuant® Running Buffer as they contain a small amount of sodium azide that could affect the results.

Materials

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.
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