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.
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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