Flow Cytometry Isotype Control Setup

1. What is Isotype Control?

An isotype control (Isotype Control) refers to an antibody that shares the same species origin, subtype, fluorescent label, dosage, and concentration as the flow cytometry antibody being used, but does not specifically bind to the target of interest. Isotype controls are typically used to eliminate background staining caused by nonspecific binding of antibodies to antigens.

2. Why Perform Isotype Control?

The binding of flow cytometry antibodies to target markers is achieved through the specific binding of the antibody's Fab end to the target marker. Leukocytes (excluding T cells) on their surfaces often express Fc receptors such as CD16, CD32, CD64, which can nonspecifically bind to the Fc end of various antibodies, leading to nonspecific staining. This phenomenon is particularly pronounced in monocytes and macrophages.

In addition to the effects of Fc receptors, in experiments involving surface staining of live cells, monocytes and macrophages can exhibit phagocytosis of antibodies or nonspecific binding with fluorescent dyes. Furthermore, in multicolor flow cytometry experiments, there can be influences from fluorescent background and drug treatments. Even if Fc receptors are blocked, in cases of poor subgrouping, isotype controls are still necessary. At such times, the use of isotype controls becomes highly important.

During experiments, relying solely on a negative control (blank control) for gating can result in some false positive results. By referencing isotype controls to distinguish between positive and negative signals, false positives caused by nonspecific binding can be eliminated.

Taking CD45+ leukocyte samples as an example, after excluding dead cells and aggregates, the fluorescence in each channel remains at a low level in the unstained, Fc receptor unblocked blank sample, as shown in the figure:

After staining with isotype controls, the background fluorescence is enhanced.

To address this strong background caused by Fc receptors, it is recommended to add an Fc receptor blocking agent, as shown in the image below:

After blocking Fc receptors and staining with isotype controls, background fluorescence is restored to a level similar to the blank sample.

3. When is Isotype Control Needed?

① For markers with clear positive and negative subpopulations, isotype controls are not necessary for gating. Examples include CD3, CD4, CD8, and other markers used for T-cell detection. For markers with unclear positive and negative subpopulations, such as activation markers like CD25, CD69, CD38, and cytokines like IFN-γ, IL-4, IL-17A, isotype controls should be used to achieve proper discrimination between positive and negative cell populations.

② In certain special cases, such as when monocytes are stained with antibodies conjugated with PE/Cyanine 7 or APC/Cyanine 7, the use of isotype control antibodies is essential. Monocytes tend to nonspecifically bind with dyes like Cyanine 7, and using isotype control antibodies helps achieve optimal results.

③ When antigen expression is unclear, it is necessary to use isotype controls to define the negative cell population.

④ For intracellular staining, due to generally low expression levels of intracellular markers, the intracellular staining process can easily lead to nonspecific binding of antigen antibodies.

4. Principles for Isotype Control Selection

► Same species origin - Mouse, Rat, Hamster

► Same subtype - IgG1, IgG2a, IgG2b, IgG3, IgM

► Same fluorescent label - FITC, PE, PerCP/Cyanine5.5, PE-Cyanine7, APC, etc.

► Same dosage and concentration (not volume-based)

Isotype controls play an irreplaceable role in flow cytometry experiments, but they also have certain limitations. Therefore, we need to determine whether isotype controls should be used in conjunction with other methods based on the specific experimental conditions. For example:

① Isotype controls are not needed for markers with clear positive and negative subpopulations; positive cell populations can be gated correctly without them.

② Isotype controls serve as a reference but should not be used directly or solely to determine positive/negative thresholds or gating. Isotype controls only reflect background fluorescence from nonspecific binding and cannot eliminate other relevant factors.

③ Since dead cells exhibit strong nonspecific staining, if there are dead cells in the sample, isotype controls along with viability dyes should be used to reduce false positives.

④ If high levels of nonspecific binding are observed when using isotype controls, it is advisable to use blocking agents to inhibit Fc receptor binding.

⑤ When using multiple fluorochromes in an experiment, fluorescence leakage can significantly affect results during analysis. Isotype controls alone cannot account for fluorescence background from leakage in other channels. In such cases, combined use of isotype controls and Fluorescence Minus One (FMO) controls may be necessary to eliminate the effects of fluorescence leakage and nonspecific binding.