B cells are antibody-producing immune cells that are used to study various infectious diseases, autoimmunity, and cancer. The in vitro culture and expansion of these cells is a useful tool for studying human immunity, B cell biology, vaccine and therapeutic antibody development, and for uncovering their potential use in cell therapies.
To help keep researchers moving forward, STEMCELL Technologies has developed products that support the complete experimental workflow; from cell sourcing and isolation to expansion and analysis (Figure 1). Here you can get some tips and tricks on how best to obtain high yields of B cells for your research.
Figure 1. Integrated Workflow for Obtaining and Culturing Human B Cells
(A) Isolate B cells from fresh human whole blood or leukopaks using EasySep™ cell separation kits or RosetteSep™ enrichment cocktails. Fresh or frozen human primary B cells can also be sourced directly from STEMCELL and immediately cultured in ImmunoCult™. (B) Expand and harvest mature B cells by culturing the isolated B cells with ImmunoCult™ Human B Cell Expansion Kit for 12 - 15 days. Passage cells every 3 - 4 days and harvest B cells for use in downstream applications. (C) Analyze B cells using STEMCELL’s antibodies, ELISA kits, and cell dyes.
*Certain products are only available in select territories.
Streamline your assays with ready-to-use human B cells isolated by positive or negative immunomagnetic selection from peripheral blood or cord blood. We help you get the cells you need with personalized service, custom products, flexible delivery times, and the option to reserve entire lots to prescreen cells for your applications*.
*Certain products are only available in select territories.
Pro Tip by Trevor Rogers, Research & Development, STEMCELL Technologies
Cell counting is an integral part of determining cell concentrations for plating in culture, determining cell viability, and assessing the results of cell isolation procedures. It is recommended to perform an initial cell count prior to cell isolation; this number can then be compared to the cell count after cell isolation to calculate cell recovery. Additionally, viable cell counts should be performed when a decrease in cell viability may be expected, for example, when working with cryopreserved cells or cells manipulated ex vivo.
Start Your Workflow by Isolating B Cells Using EasySep™
For experiments that require a high purity of B cells, it is necessary to enrich the samples before progressing to cell culture or expansion. EasySep™ is a fast, easy, and column-free immunomagnetic cell separation system for isolating highly purified cells. Below is a representative flow cytometry plot showing the content of pan-B cells in a sample enriched with EasySep™ Human Pan-B Cell Enrichment Kit.
Figure 2. EasySep™ Human Pan-B Cell Enrichment Kit Isolates Pan-B Cells with High Purity
Representative flow cytometry plots gated on viable cells isolated with EasySep™ Human Pan-B Cell Enrichment Kit. Starting with nucleated cells, the pan-B cell [Lineage (CD4, CD8, CD14, CD16, CD56) negative, CD19+ and CD19-CD43+] content of the enriched fraction typically ranges from 90 - 99%.
EasySep™ enables us to isolate B cells from the spleen really fast with good viability. That is what we need. You don’t have to stand in front of a column and wait for it to drip. The viability of the cells that you recover tends to be higher just because it is so fast.
If you're working with unique or rare B cell subsets in your research but can't find a readily available cell separation kit, explore our custom cell separation products for solutions tailored to your exact research needs. Contact Product & Scientific Support at email@example.com to discuss your specific cell separation needs or connect with us on LiveChat.
Culture Human B Cells with ImmunoCult™
B cells sourced from STEMCELL or isolated with EasySep™ are highly purified and immediately ready for cell culture. Achieving robust expansion of B cells in culture typically requires the addition of serum or feeder cells. Addition of serum can improve performance, albeit with high lot-to-lot variability in the composition of the serum and the risk of contamination by adventitious agents. To minimize this variability, it is important to use serum-free medium and supplements, such as ImmunoCult™-XF B Cell Base Medium and ImmunoCult™-ACF Human B Cell Expansion Supplement. To achieve the most optimal expansion, use the ImmunoCult™ Human B Cell Expansion Kit, which is designed to give high cell yield and frequency without the use of serum, specialized cultureware, or feeder cells.
Figure 3. Culture B Cells with ImmunoCult™ Human B Cell Expansion Kit
B cells isolated from human PBMCs (leukopak) using EasySep™ Human Pan-B Cell Enrichment Kit were seeded at 1 x 105 cells/well in 24-well tissue culture plates and cultured using ImmunoCult™ Human B Cell Expansion Kit. The cells were passaged every 3 - 4 days. (A) Fold expansion of viable cells is shown for n = 12 donors, with bars representing the mean and 95% confidence level (range: 38- to 1190-fold at day 14 ± 1 day). (B) Expression of CD138 and CD20 was analyzed by flow cytometry at each timepoint (data represent % positive viable cells; mean ± 1 SD). The observed changes indicate maturation of B cells to plasma cells/blasts.
Pro Tips by Hitesh Arora, Research & Development, STEMCELL Technologies
To ensure high cell viability and expansion, passage cells every 3 - 4 days and avoid letting cultures become over-confluent
To avoid introducing error in cell counts due to local variation in cell density, cells should be well-mixed before counting
Do not disturb the cells while in the incubator between passaging steps; this can affect B cell clustering
Always perform the experiment in sterile conditions
Antibody development and screening
Studies on human immunity and infectious diseases
Research in understanding B cell biology, signaling, differentiation, and development
Analyze and Characterize B Cells
Expanded B cells can be used in downstream applications and may be analyzed and characterized using techniques such as flow cytometry, ELISA, microscopy etc. Figure 4 below shows how B cells undergo morphological changes as they mature.
Evaluate B Cell Culture
Figure 4. Robust Growth of Human Pan-B Cells Cultured with ImmunoCult™ Human B Cell Expansion Kit
Pan-B cells isolated from human PBMCs (leukopaks) were seeded at 1 x 105 cells/well in a 24-well tissue culture plate with ImmunoCult™ Human B Cell Expansion Kit. The cells were passaged on day 4 after seeding and imaged at (A,C) 20X, (B) 2X, or (D) 40X objective, 6 days after seeding. At (A) Day 0, B cells appear small and not clumped together; however, at (B, C, D) Day 6, B cells form large colonies and increase in size, suggesting they are undergoing activation and maturation.
Viability and functionality
Antibody production and secretion
Assess Phenotype of Mature B Cells
Figure 5. Expression of Activation and Maturation Markers on Human B Cells Cultured with ImmunoCult™ Human B Cell Expansion Kit
B cells isolated from human PBMCs (leukopak) using EasySep™ Human Pan-B Enrichment Kit were seeded at 1 x 10^5 cells/well in 24-well tissue culture plates and cultured using ImmunoCult™ Human B Cell Expansion Kit. The cells were passaged every 3 - 4 days. Expression of CD19 (A, B), CD20 (C, D), CD86 (E, F) and CD138 (G, H) was analyzed by flow cytometry, with profiles shown at Day 0 (upper panel) and Day 11 (lower panel). The observed changes indicate activation (CD86) and maturation (CD20, CD138) of B cells to plasma cells/blasts.
Pro Tips by Mourad Van Hove, Scientific Support, STEMCELL Technologies
To ensure success when you characterize your B cells using flow cytometry, there are a number of factors to consider and incorporate into your staining and gating strategy:
Compensate for spectral overlap
Use appropriate gating controls
Exclude debris and non-viable cells
Exclude doublets or multiplets
Stain with a pan-leukocyte marker (e.g. CD45)
If you need antibodies for your B cell characterization, you can view our inventory via the link below:
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