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.
For the isolation of human PBMCs refer to the protocol "Isolation of mononuclear cells from human peripheral blood by density gradient centrifugation".
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.
For B cell isolation various reagents and kits are available according to the kind of isolation.
Download the quick guide and find a comprehensive overview of all B cell markers and products for convenient isolation and analysis.
This protocol is optimized for the activation and expansion of B cells, using the Human CD40-Ligand Multimer Kit (component of the B Cell Expansion Kit, human). B cells can either be expanded in low density (0.15×10⁶ cells/mL) or high density (1×10⁶ cells/mL) cell culture.
Table 1: Culture plate sizes and amount of medium for different cell sizes.
|Total cell number|
|Total cell number|
|Final medium volume||Culture plate|
|0.5×106||0.075×106||0.5 mL||48 well|
|1.0×106||0.15×106||1 mL||24 well|
|2.0×106||0.3×106||2 mL||12 well|
|4.0×106||0.6×106||4 mL||6 well|
PBS/EDTA: Phosphate-buffered saline (PBS), pH 7.2, with 2 mM EDTA.
▲Note: EDTA can be replaced by other supplements, such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD).
50 mL conical centrifuge tubes
MACSQuant® Analyzer 10 (# 130-096-343) or other flow cytometers equipped with violet (405 nm) and red (635 nm) lasers able to discriminate FITC, PE, and APC fluorescence.
MACS Chill 96 Rack (# 130-094-459) when using MACSQuant Analyzer 10
MACSQuant Calibration Beads (# 130-093-607) when using MACSQuant Analyzer 10
MACS Comp Bead Kit, anti-REA (# 130-104-693) for optimal compensation of the fluorescence spillover from fluorochrome-conjugated antibodies
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