Yes, if you want to transfect mouse B cells, we recommend to stimulate the cells before. The information is also available in the mouse B cell nucleofection protocol. Our R&D has used following combination : RPMI1640 [Lonza; Cat. No. 12-167F] supplemented with 10% FCS, 2 mM UltraGlutamine I [Lonza, Cat. No. 17-605E/U1],
50 µM ß-mercaptoethanol, 1% ITS [Sigma] and LPS (50 µg/ml) if desired . General points to consider when you work with mouse B cell stimulation protocols:
The media you grow your purified cells in can have a huge impact on your experimental outcomes. Usually you grow B cells in 10% FBS in RPMI. Be careful! The serum components can alter B cell responses in vitro. Before starting your experiments, test multiple different serum lots on your primary B cells and determine how they affect the outcomes of your particular assays. Once you have found a suitable lot of FBS, you should stock up and use the same lot number for all of your experiments.
Make sure you make your media fresh. Media components, like glutamine, are critical for cellular growth and proliferation but can deteriorate quickly in the media. Alternatively, use more stable analogues like GlutaMAX.
Many labs include beta-mercaptoethanol as a reducing agent in their cultures. Although the mechanism is not understood, beta-mercaptoethanol improves cell viability. You might want to consider adding it.
In culture, primary murine B cells die quickly without stimulation; they need pro-survival signals to avoid apoptosis. You can use different types of stimulation depending on the desired outcome. Three commonly used stimulants are:
- lipopolysaccharide (LPS)
- anti-IgM antibodies or
- a combination of CD40 ligand/anti-CD40 antibodies and IL-4
LPS is a strong mitogen in primary B cells. However, this stimulation occurs through crosslinking of TLR4, which is not expressed on human B cells. You can also use LPS in combination with co-stimulation by IL-4 and/or CD40 to stimulate a more biologically-relevant response.
IgM stimulation mimics a T cell-independent reaction by massive crosslinking of the B cell receptor. However without co-stimulation these cells usually do not proliferate. This stimulation is great for studying B cell receptor signaling!
Stimulation through CD40 and IL-4 mimics a T cell-dependent immune response and will induce proliferation and differentiation, closely mimicking an actual B cell immune response.