Why Cell Stimulation Matters in Flow Cytometry
Many flow cytometry users are happy to start an experiment with a general protocol and a question about their specimen: Will my cells make more cytokines or express more markers after activation?
In a healthy person, immune cells like B cells, T cells, and macrophages typically surveil the body for abnormal cells or infectious agents. These cells don’t fire up their inflammatory toolbox unless they recognize one of these foreign entities. The potent inflammatory mediators activated during these responses include cytokines, free radicals, prostaglandins, and clotting factors, which must be tightly regulated to avoid wreaking havoc on healthy tissue. This exquisitely controlled activation of inflammatory molecules means that when you look for them in cells by flow cytometry, they may be very difficult to detect.
What Is Cell Stimulation and When Do You Need It?
Understanding the Role of Baseline Controls
Baseline (unstimulated) controls show what your cells look like at rest and provide the reference point for interpreting stimulated samples. They help distinguish true biological responses from background signal, nonspecific staining, or unintended activation during handling. In some studies, baseline expression alone can be informative, revealing differences in cell state, depending on your goals.
Types of Cell Stimulation Used in Flow Cytometry
Most flow cytometry staining protocols start with isolating your cells of interest and manipulating them in culture for staining with immunofluorescent antibodies. These protocols typically include a step in which cells are stimulated with a mitogen, a chemical that can broadly and nonspecifically activate your cells. You may also include a separate stimulation with a specific molecule or compound of interest, such as the protein subunit used in prior activations.
Mitogenic Stimulation
Mitogenic stimulation is a fast and reliable way to make sure your cells are functional. They will produce cytokines and express cell surface markers in response to stimulation, so even if your experimental stimulation doesn’t work, you can be assured that your cells are viable and functional and your staining protocol worked correctly.
Specific Stimulation (e.g., Antigen- or Drug-Specific)
Specific stimulation allows you to screen drugs or biologics to determine which candidates can activate specific cells in a desirable way or identify potentially dangerous responses in vitro before further testing in animal models or patients.
Specific stimulation allows you to test how your cells respond in vitro, which is critical to evaluating vaccine recall responses from animal models and patient samples.
Questions to Ask When Designing Your Assay
What Do You Want to Know About the Cells?
Do you want to see the cells at rest, or do you want to measure how much they can respond to a specific stimulus? No matter what type of protocol you use, you will always need to measure the production of inflammatory molecules in unstimulated cells as a control.
How Specific Should Your Stimulus Be?
Immunologists use a variety of different mitogenic stimuli to induce broad and non-specific responses by immune cells, and this can be useful for studies to understand the characteristics of different immune cells. In contrast, vaccine studies need to look at the specificity of an immune response, which requires in vitro stimulation assays with the same components as used for vaccine trials.
Why It’s Critical for Preclinical and Immunology Studies
Cell stimulation is a fundamental step and key control for flow cytometry experiments. Be sure to consider all the essential cell stimulation scenarios you will need as you plan your next flow cytometry experiment. These basic questions should help you discern whether or not you need to stimulate your cells in vitro for your flow cytometry assays.