The Care and Keeping of Primary Murine B Cells

So you want to work with mouse B cells?

Primary murine B cells are a difficult, yet fascinating system to work with and can help deepen your understanding of an immunological system. You can study many things with primary B cells, including:

• immune activation
• antibody production
• cell-cell interactions between immune cells and
• immune phenotype

These cells can be fickle to work with, but here are a few tips to help you decide what matters when culturing primary murine B cells in your lab.

Separation matters

While you can isolate B cells from mouse bone marrow and lymph nodes, spleen offers the best yield of mature B cells for stimulation.

Two common ways to separate B cells are by positive selection for CD19 (which is only expressed on B cells) or negative selection for CD43 (which is expressed on most leukocytes except for mature B cells).

Negative selection has the distinct advantage of leaving your B cells unstimulated and is the most commonly used selection for activation assays. However, CD43 can also be expressed on plasma cells, so information about antibody-producing cells might be lost.

Media matters

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 ?-mercaptoethanol as a reducing agent in their cultures. Although the mechanism is not understood, ?-mercaptoethanol improves cell viability. You might want to consider adding it.

Stimulation matters

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.

Limitations matter

Genetic manipulation of primary B cells is notoriously difficult and a problem the entire field is working to address. Proceed with caution if your experiments require knockdowns or overexpression to address your hypothesis.

If you’re concerned about the differences between mouse and human B cells and have access to human tonsils or peripheral blood, human B cell culture requires only a few changes in selection and stimulation.

With these guidelines, you should be well on your way to bringing B cell biology into your own lab.

Have you worked with primary muring B cells? What tips or questions do you have?