Importance of Isotype Controls in Cytometry Experiments

In flow cytometry experiments, the accuracy and reliability of the results depend largely on correctly interpreting the fluorescent signal. It is crucial to distinguish between the specific signal, which originates from the binding of the antibody to the antigen of interest, and the nonspecific or background signal. To achieve this, the use of appropriate controls is essential. Among these, isotypic controls play a fundamental role.

What are isotypic controls?

Isotypic controls are antibodies that do not have specificity for the target antigen but share the same characteristics as the specific antibody used in the experiment. These include:

-The same immunoglobulin class and subclass (isotype).

-The equals light chain.

-The same fluorochrome conjugation.

 Why are they important in flow cytometry?

Isotypic controls allow the evaluation of non-specific binding of antibodies in a flow cytometry experiment. This is crucial to correctly interpret the results and avoid erroneous conclusions.

Main functions of isotypic controls:

1. Distinguish specific signal from background: By comparing the fluorescence of the isotypic control with that of the specific antibody, one can determine how much of the observed signal is actually due to antigen binding and not to nonspecific interactions.
2. Optimize antibody concentration: Help adjust the amount of antibody used in the experiment to minimize nonspecific binding without compromising antigen detection.
3. Control cellular autofluorescence: Some cells may naturally emit fluorescence, which can interfere with antigen detection. Isotypic control allows this effect to be identified.
4. Improve interpretation of results: Especially in samples with cells with high Fc-receptor expression (such as macrophages and dendritic cells), isotypic controls help to assess whether antibody binding is due to interactions with these receptors and not to the target antigen.

How are isotypic controls used in an experiment?

1. We include isotypic antibodies labeled with the same fluorochrome as the specific antibodies in the cytometry panel
2. We incubate them with a fraction of the sample under conditions identical to those of the antibody of interest.
3. Compare the fluorescence between the sample stained with the specific antibody and the sample stained with the isotypic control.

Limitations of isotypic controls

While useful tools, isotypic controls have some limitations:

-They do not always accurately reflect non-specific binding of the specific antibody.

-I do not consider antibody aggregation effects or variations in epitope accessibility.

-In some cases, the use of an unstained cell control or a FMO (Fluorescence Minus One) control may provide more accurate information about the specific signal.

Conclusion

For more accurate results and a better understanding of how isotypic controls can improve your flow cytometry experiments, feel free to consult our range of available products.