Introduction of Flow Cytometry | Summary of Flow Cytometry Staining Principles

In previous issues, we extensively discussed the principles of multicolor flow cytometry panel design. Today, let's summarize and review together.

The 6 Key Principles of Optimal Antibody Combinations

1. Choose strong fluorescent dyes for weakly expressed antigens and weak fluorescent dyes for strongly expressed antigens (traditional rule).

2. Place weakly expressed antigens in channels that do not interfere with other channels, while placing strongly expressed antigens in channels that do not interfere with them.

3. Mutually exclusive antigens are allowed to have spillover.

4. Spillover should be avoided for antigens that are co-expressed.

5. Allowing fluorescence spillover from descendant to progenitor, but not the reverse.

6. Aim to reduce the complexity of data analysis.

Flow Cytometry Panel Design Principles (I) — Matching Intensity: Matching weakly expressed antigens with strong fluorescent dyes, and strongly expressed antigens with weak fluorescent dyes. When pairing, it's crucial to determine antigen density at the cellular subset level, which can also vary due to activation states. The expression levels of markers can be referenced from relevant literature or data, while the intensity of fluorescent dyes can be determined using tables or staining indices.

Figure 1. Types of Fluorophores

Flow Cytometry Panel Design Principles (II) — Placing Weakly Expressed Antigens in Non-Interfering Channels: Place weakly expressed antigens in channels where other channels do not interfere. A non-interfering channel means other channels do not cause interference, such as the FITC channel. For instance, in multicolor analysis of human peripheral blood, CD45 is highly expressed and can easily interfere with the detection of weakly expressed antigens. In such cases, CD45 can be paired with a fluorophore that does not interfere with other channels, such as Elab Fluor® Violet 450 (Pacific Blue channel), thus avoiding interference with dyes excited by 488 nm or 633 nm lasers.

Why should fluorescence interference be avoided? This is because adjusting compensation can only eliminate fluorescence background, while the decreased fluorescence sensitivity in the interfered channel cannot be restored through compensation adjustments. Therefore, when designing panels, it's crucial to minimize mutual fluorescence interference to obtain more accurate results.

Figure 2. Impact of Fluorescence Interference

Flow Cytometry Panel Design Principles (III) — Allowing Fluorescence Spillover for Mutually Exclusive Antigens: When designing panels, consider the relationship between antigens. If antigens have a mutually exclusive relationship, you can use highly interfering fluorescent combinations. Mutually exclusive antigens refer to two antigens that do not express simultaneously on one type of cell. For example, in CD3+ T cells, there is a subset of cells expressing CD4 but not CD8, making CD4 and CD8 mutually exclusive antigens. In this case, CD4 and CD8 can be paired with FITC and PE, respectively, which are two fluorescent dyes with significant interference.

Figure 3. CD4 and CD8 are mutually exclusive antigens

Flow Cytometry Panel Design Principles (IV) — Avoiding Fluorescence Spillover for Co-Expressed Antigens: If antigens are co-expressed, avoid fluorescence spillover. Co-expressed antigens refer to antigens that are simultaneously expressed, such as CD25 and Foxp3 in Treg cells. When analyzing double-positive cells on flow cytometry plots, CD25 and Foxp3 can be paired with APC and PE, respectively, which are fluorescent dyes with minimal interference.

Figure 4. CD25 and Foxp3 are co-expressed antigens in Treg cells

Flow Cytometry Panel Design Principles (V) — Allowing Descendant to Progenitor Fluorescence Spillover: If antigens have a descendant-to-progenitor relationship, allow fluorescence spillover from the descendant to the progenitor antigen, while avoiding spillover from the progenitor to the descendant. For instance, in CD4+ T cells, since all CD4+ T cells express CD3, CD3 can be paired with PE as the progenitor, and CD4 can be paired with FITC as the descendant. Here, CD4 is in a non-interfering channel, facilitating compensation adjustments and benefiting data analysis.

Figure 5. Results of CD3-FITC and CD4-PE dual staining

There are many principles to consider in panel design, and often we cannot completely adhere to each one. However, by applying these principles as much as possible, we can minimize the complexity of data analysis.

When designing panels, we often say: "There's no best, only better." Panel optimization is a flexible process. Elabscience® offers flow antibodies labeled with up to 18 different fluorochromes, suitable for various experimental setups. We provide free panel design and data analysis services, offering comprehensive flow cytometry solutions.