Are you looking for ways to monitor immune responses in your experiments? Two commonly used techniques are enzyme-linked immunospot (ELISPOT) and flow cytometry. While both methods provide valuable information, they differ in their approach, outputs, and areas of applications. This post aims to compare the two techniques and to help you decide which one to use for your specific needs.
Methodology
ELISPOT assays and flow cytometry both provide information on cells' ability to produce and secrete specific proteins, such as cytokines or antibodies, in response to a stimulus.
ELISPOT assays typically involve incubating cells in microwell plates coated with capture antibodies, stimulating cells with antigens or mitogens, and then detecting secreted cytokines using enzyme-conjugated detection antibodies. The enzymatic reaction results in the formation of spots in the wells, indicating the cells' ability to secrete cytokines. For example, the number of interferon-gamma (IFN-γ) spots represents the number of cells that secreted this cytokine.
Flow Cytometry, on the other hand, involves labeling cells with fluorescent antibodies directed against specific cell surface markers or intracellular antigens. As cells flow in a stream past a laser detector, the antibodies bind and emit a signal that indicates the cells' characteristics, including size, granularity, and marker expression.
Output Data
ELISPOT assays generate qualitative results, i.e., the number of spots produced in the wells, while flow cytometry provides quantitative data, i.e., the percentage of cells with a particular marker expression. ELISPOT assays provide information about the frequency of cells secreting specific cytokines or antibodies, while flow cytometry can characterize and sort cells based on multiple parameters.
Applications
ELISPOT assays are commonly used to measure T cell responses against infectious agents, tumors, or autoantigens. They are also used to monitor vaccine efficacy or to evaluate immune responses in patients with immune-related diseases. ELISPOT assays are less sensitive to rare cell populations and do not require single-cell isolation often needed in flow cytometry. ELISPOT assays can also be performed on peripheral blood mononuclear cells (PBMCs), which represent a heterogeneous mix of immune cells.
Flow cytometry is a versatile technique used in multiple fields of biology and medicine to study cellular properties, including cell cycle, apoptosis, proliferation, and differentiation. Flow cytometry is also used in immunology to investigate immune cells' activation, differentiation, and function. Flow cytometry is better suited to analyze rare cell populations and can be used to sort cells with specific characteristics.
Pros and Cons
ELISPOT assays are generally easier to perform and have fewer steps and lower costs than flow cytometry. ELISPOT assays are relatively robust and provide consistent results across different laboratories. However, ELISPOT assays have several limitations, including lower sensitivity, lower dynamic range, and variable background noise.
Flow cytometry, although more challenging to perform, provides more advanced features such as multi-parameter cell analysis and sorting. However, flow cytometry requires extensive training, expensive instrumentation, and careful design of experiments. Additionally, flow cytometry results can be affected by variations in sample processing and acquisition.
Conclusion
In summary, ELISPOT assays are advantageous for studying cytokine or antibody production by cells at a population level, while flow cytometry is better suited to study cells' properties at the single-cell level. In general, ELISPOT assays are cheaper, easier and faster to perform, but less sensitive and less versatile than flow cytometry. As always, the choice between ELISPOT and flow cytometry depends on the scientific question, resources, and experimental design.
References:
- Janetzki et al. Guidelines for the automated evaluation of Elispot assays. Nat Protoc. 2015 Nov;10(11):1098-115.
- Cossarizza et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies. Eur J Immunol. 2017 Jan;47(10):1584-797.