Introduction of Flow Cytometry丨 Principles of Flow Cytometry Staining Lesson 1 - Balance Antigen Density and Fluorochrome Brightness

Flow cytometry staining can indeed be challenging, but with the right understanding of principles, you can navigate through it more smoothly. Let's break down the interpretation of the first principle of flow cytometry staining: Balance Antigen Density and fluorochrome Brightness.

The first rule of multicolor flow cytometry staining: Strongly expressed antigens should be paired with weak fluorochrome,weakly expressed antigens should be paired with strong fluorochrome. This principle emphasizes the importance of matching the intensity of the target marker expression with the intensity of the fluorochrome.

When strongly expressed antigens are paired with strong fluorochrome and weakly expressed antigens are paired with weak fluorochrome, there can be interference between the strong and weak fluorochrome. In such cases, the signal from weakly expressed antigens may be obscured by the fluorochrome interference. It's similar to how during the day, when the sun is out, we cannot see the stars in the sky. It's not that the stars have disappeared; rather, the intense sunlight overwhelms the light from the stars.

As illustrated in the following scenarios:

· For CD4, which is strongly expressed, choosing either a weak fluorochrome FITC or a strong fluorochrome PE doesn't significantly impact the results.

· For CD25, which is weakly expressed, when paired with weak fluorochrome PerCP/Cy5.5 and CD4-PE, positive cell groups of the CD25 are obscured. However, when CD25 is paired with strong fluorochrome APC, positive cell groups can be obvious to observe.

■ CD4-PE ■ CD25-PerCP/Cy5.5 ■ Foxp3-FITC

■ CD4-FITC ■ CD25-APC ■ Foxp3-PE

The expression level of the target marker for detection should be determined by referring to relevant literature or resources. It's important to note that antigen density is always determined at the level of cell subsets, and antigen density can also vary due to activation status.

Now, let's identify which fluorochromes are considered strong or weak. Don't worry, I've already organized them for you. Fluorochrome brightness is categorized into four types: very bright, bright, moderate, and dim, as shown in the diagram below:

If you're using a fluorochrome that's not listed in the table, you can determine its brightness by calculating the SI (staining index). The staining index can help assess the brightness of the fluorochrome.

Staining Index (SI) = D/W

Where:

D = Difference between the mean fluorochrome intensity (MFI) of the positive population and the MFI of the negative population.

W = Width of the negative population.

The higher the staining index, the brighter the fluorochrome of the dye.

That wraps up our interpretation of the "intensity matching" principle in flow cytometry staining. If you have any questions about flow cytometry staining, feel free to leave them in the comments. Stay tuned for more insightful content on flow cytometry staining in the Introduction of Flow Cytometry series!