Antibodies 101: Designing Your First Flow Panel

By Guest Blogger

Guest Blogger May 28, 2024

When analyzing your cells using flow cytometry, you are typically measuring the presence or absence of certain markers on the surface or the inside of your cells. While proteins themselves can emit intrinsic fluorescence when excited with ultraviolet (UV) light, they do so via aromatic amino acids found in all proteins, so you can't distinguish the different proteins from each other. To distinguish different target proteins in your flow analysis, you'll need to use fluorophores that attach to the target. When you design a flow experiment, you'll need to pick a fluorophore for each target and ensure the fluorophores, together, are able to give you the appropriate readout.

A common method of staining proteins is to fuse them with a fluorescent reporter, such as GFP or mCherry, through genetic modification. As your protein of interest will only occur in combination with that reporter, you can infer its presence through detecting the reporter's fluorescence. Another way to stain proteins is through treating cells with fluorophore-conjugated antibodies that attach to a specific target. By using a mix of different antibody-fluorophore combinations for different markers of interest, you can separately detect these markers in your analysis. Lastly, there are fixable dye stains that react with free amines on and inside the cells and are thus typically used for distinguishing live from dead cells. The combination of antibody-fluorophores, fluorescent reporters, and/or dye stains used to in your flow cytometry experiment are known as your "panel". In this blog post, we'll discuss the principles and process of designing a flow panel.

Principles of Excitation and Emission

Before we design your first panel, we will introduce some background on the color spectrum and the principles of excitation and emission. The visible color spectrum is located in a range of wavelengths of about 380–700 nm. Photons of lower wavelengths are higher in energy, while photons of higher wavelengths are lower in energy. As such, wavelengths below 380 nm lie within the UV spectrum, while wavelengths over 700 nm make up the infrared (IR) spectrum.

When photons are absorbed by matter, they promote electrons within atoms to a higher energy state. This process is called excitation. After a short period, the electrons revert to a lower energy state, whereby a photon is emitted. This step is called emission. During excitation, some of the photon's energy is lost, so the energy of an emitted photon is lower than the energy of the absorbed photon. Correspondingly, wavelengths of emitted photons are higher than those of absorbed photons.

When photons excite electrons of a fluorophore, they can do so at a range of wavelengths, rather than at just one defined wavelength. Likewise, the emitted photons also appear within a range of wavelengths. For that reason, we speak of excitation and emission spectra.

Both spectra possess clear maxima where excitation and emission are the most efficient (i.e., happen most of the time). Figure 1 shows a graph from the BD® Spectrum Viewer, outlining a few commonly used fluorophores and their emission spectra. Some fluorophores carry their emission peaks within their names, for example, BV421 and RB545.

Fluorophores can also come in combination of two conjugated single fluorophores, like APC-Cy7. Conjugating Cy7 to APC results in a higher-wavelength emission spectrum compared to APC. This happens due to a process called Fluorescent Resonance Energy Transfer (FRET).

Graph of excitation/emission (y axis) of ten colors by wavelenth (x axis), showing each color's excitation/emission peak. Although each peak is distinct. there is considerable overlap of the shoulders in many of the colors.

Figure 1: Emission spectra and respective colors of a few commonly used fluorophores. Each fluorophore has a defined emission maximum. Note, you can also use the tool to display excitation spectra, which I excluded here for better visibility. Source: https://www.bdbiosciences.com/en-us/resources/bd-spectrum-viewer.

Brightness

Apart from the excitation and emission wavelengths, it is also important to know the brightness of individual fluorophores. Table 1 describes the brightness of a few commonly used fluorophores. A fluorophore's brightness refers to the signal strength of the emitted photons detected by the cytometer. Brighter fluorophores have a stronger signal.

Table 1: Degrees of brightness for a few commonly used fluorophores in relation to the laser type. Table adapted from: https://emea.bd.com/panel-design/en/flow-cytometry-fluochrome-brightness-spillover

Excitation Laser Color	Very Bright	Bright	Moderate	Dim
Ultraviolet (355 nm)		BD Horizon™ BUV737	BD Horizon™ BUV395
Violet (405 nm)	BD Horizon™ BV421 BD Horizon™ BV650 BD Horizon™ BV711	BD Horizon™ BV605 BD Horizon™ BV786	BD Horizon™ BV510	BD Horizon™ V450 BD Horizon™V500
Blue (488 nm)	BD Horizon™ BB515 BD Horizon™ PE-CF594 Pe-CyBD™5	PE PE-Cy™7	FITC Alexa Fluor 488 PerCP-CY5.5	PerCP
Yellow/Green (561 nm)	PE BD Horizon™ PE-CF594 PE-Cy5 PE-Cy7
Red (640 nm)		APC Alexa Fluor 647		Alexa Fluor 700 APC-H7 APC-Cy7

Many factors can influence the level of brightness beyond the fluorophore itself, like laser type and power, acquisition voltage, the concentration of the fluorophore, and whether that fluorophore appears along or in conjugation to another fluorophore. The brighter a fluorophore, the better the signal-to-noise ratio, and the better the detection of the target marker.

Looking at Table 1, we can recognize a couple of fluorophores from Figure 1. For example, the chart tells us that PE and PE-Cy7 are "very bright" when they are excited by the yellow/green laser but only "bright" for the blue laser.

To understand why that is, let's look at Figure 2. The blue laser excites at a wavelength of 488 nm (Table 1), which is near a local maximum of PE's excitation spectrum (dotted line, Figure 2). The yellow/green laser, however, excites at 561nm, which is close to the global maximum of PE's excitation spectrum where the excitation efficiency is much greater. As a result, more photons are absorbed and then emitted, which creates a relatively stronger—or brighter—signal.

Figure 2: Excitation and emission spectra for PE. The excitation spectrum shows two maxima, one local maximum at just under 500 nm and a global maximum at around 560 nm, leading to different levels of brightness, depending on the laser light's wavelength. Source: https://www.bdbiosciences.com/en-us/resources/bd-spectrum-viewer.

Another example

APC is a "bright" fluorophore when excited by the red laser. When conjugated to Cy7, however, (APC-Cy7) the brightness changes to "dim" for the same laser type. Knowing that the red laser excites at 640 nm (Table 1), while comparing the two fluorophore's excitation spectra around 640 nm (Figure 3), we see that APC-Cy7 is excited at a lower efficiency than APC. While APC's global excitation maximum lies around 650 nm, APC-Cy7's global excitation maximum is about 750 nm. If we were to use a laser that excites at 750 nm, APC-Cy7 would show a much brighter signal, while APC would show no signal at all, as it cannot be excited at this wavelength.

Figure 3: Excitation and emission spectra for APC and APC-Cy7. While the excitation spectra of both fluorophores show efficient absorption at 650 nm, APC-Cy7's optimal excitation happens at higher wavelengths of about 750 nm. Source: https://www.bdbiosciences.com/en-us/resources/bd-spectrum-viewer.

But what is the real-life advantage of a brighter fluorophore?

Strong brightness makes it easier to distinguish a signal from noise; therefore, it is recommended to use brighter fluorophores for markers that are relatively lower in abundance. In contrast, when you expect a marker to be highly abundant, it is fine to use a dimmer fluorophore. However, these aren't strict rules and in practice the differences in brightness levels are often not that great. When thinking of terms like "very bright" vs. "dim" you might get the impression of a candlelight vs. a floodlight. For fluorophore brightness levels, these differences are much more subtle (think of brightness levels on your smartphone or computer screen), and you should be able to properly stain many markers with any fluorophore.

Bleeding

Before we start building your first panel, we'll talk about one last principle of color emission.

As you can see in Figure 1, emission spectra of neighboring colors can and do overlap. For example, APC overlaps noticeably with both BV605 and Alexa Fluor 700. It also overlaps with a few other fluorophores, although to a much lower degree. The phenomenon of spectral overlap in flow cytometry is commonly referred to as bleeding. When two fluorophores bleed into each other, it means that the cytometer could detect a certain photon to be emitted by either of the two. As a result, inaccuracies in the detection of fluorophores (and the markers they label) can occur.

As I mentioned previously, you can also use fluorescent reporters, such as GFP, and fluorophore-conjugated antibodies together in the same color panel. In this case, make sure to compare the excitation/emission spectra of your reporter with the ones from your fluorophore stains and see whether and to what extent they overlap. Figure 4 compares the spectra of enhanced GFP (eGFP) and FITC, the latter being a commonly used fluorophore for antibody-conjugated stains. Even though the two compounds are completely different in nature (eGFP being a protein and FITC an organic molecule), their bleed is so strong that these spectra almost overlap. Hence, they should not be used within the same panel.

Excitation and emission spectra for eGFP and FITC, with both colors peaking around 525 nm. It is difficult to distinguish the two peaks.

Figure 4: Excitation and emission spectra for eGFP and FITC. As these stains' spectra virtually overlap, you cannot use them together in the same panel. Source: https://www.bdbiosciences.com/en-us/resources/bd-spectrum-viewer.

To make life easier, always try to use fluorophores with distant excitation/emission spectra to avoid bleeding as much as possible. However, when you need to stain for multiple markers, some bleeding is often unavoidable. But don't worry, there are strategies to resolve this issue. Make sure to check out our blog post on color compensation to learn more about them. Alright, let's build your first panel!

Designing a Panel

In this example, I will describe a basic panel (Table 2) for a hypothetical experiment in the lab. Let's say I am culturing HEK 293T cells in vitro. I transduced the cultured cells with a virus that encodes a transgenic protein for the HEK 293T cells to express: CD45. Upon expression, CD45 is incorporated into the plasma membrane and can be detected through a cell surface stain. The viral vector also encodes for eGFP, which serves as a transduction reporter. Note that eGFP is not fused to CD45, but is separately expressed. (The reason for having a separately expressed transduction reporter is that not every transduced cell will necessarily manage to express CD45. Thus, if transduction efficiency is much higher than CD45 expression, this could, for example, point at issues with the double-expression vector.) In our flow experiment, we'll want to identify CD45⁺ cells (CD45 expression) and assess our transduction efficiency (eGFP expression).

The first stain in Table 2 is a standard and should always be included—the live/dead stain. This stain is in fact not created through the actual APC-Cy7 fluorophore but displays an emission spectrum that overlaps with APC-Cy7. Therefore, you can detect this stain through the APC-Cy7 parameter in the cytometer's software. It also means we should exclude APC-Cy7 from the rest of our panel. Besides some background cell death constantly happening in cell culture, the transduction process itself also leads to some cell death. As a result, there is a high abundance of positively stained dead cells present and it is fine to use a dim color, like APC-Cy7.

Next comes our transduction reporter, eGFP. As eGFP emits fluorescence by itself and does not need to be bound through a separate fluorophore, we have to tell the cytometer which channel to use to detect eGFP. As you saw in Figure 4, eGFP strongly overlaps with FITC, hence reading out the FITC channel in the cytometer's software (without using the actual FITC fluorophore in our panel) will allow us to detect the eGFP signal. We will now exclude FITC from the rest of our panel.

Lastly, we want to detect the expression of our protein of interest: CD45. I am using a conjugated antibody to detect CD45, and I'll need to choose the fluorophore my antibody is conjugated to. Since CD45 is a common marker, finding an antibody already conjugated to my fluorophore of choice will not be difficult.

I chose BV421 for the following two reasons: 1) The emission spectrum of BV421 is far enough from the other fluorophores to avoid bleeding and 2) BV421 is a very bright fluorophore, which makes it easier to detect the transgenic protein. We don't know yet how abundantly CD45 will be expressed on the transduced cells, so it's better to use a brighter fluorophore in case the CD45 expression turns out to be weak.

Table 2: A simple staining panel to detect live, transduced, and CD45-expressing cells.

Marker	Fluorophore detection
Live	APC-Cy7 channel
eGFP	FITC channel
CD45	BV421

Emission spectra of BV421, eGFP, and APC-Cy&. BV421 and eGFP have distinct peaks at ~425 and ~525 nm, respectively, with minimal overlap at their tails (~480 nm). APC-Cy7 has a strong peak at ~775 nm and does not overlap with the other two colors.

Figure 6: Emission spectra of the fluorophores used in our panel (Table 1).

Conclusion

You made it! In this post, you learned about the principles behind fluorophores and their colors for used in flow cytometry. Let's briefly go through each section of this article again. In the beginning, we talked about the relationship between a photon's wavelength and energy, how photons can be absorbed and emitted by matter, and how this process creates visible colors. Next, you learned about how fluorophores can be classified into different levels of brightness and their relation to the fluorophore's excitation efficiency, and when it might be better to use a brighter fluorophore. Furthermore, you discovered the concept of bleeding, i.e. spectral overlap, and why you should try to avoid or reduce it whenever possible. Finally, we designed a simple panel to analyze transduced HEK 293T cells. Here, we looked at both the transduction efficiency and the expression of the transduced protein. I hope that helps you as you begin to design your first flow panels!

Paul Heisig is a Research Associate in the lab of Arlene Sharpe at Harvard Medical School. His projects include investigating negative regulators of T cells and cytokine signaling in tumor immunity. When he's not in the lab, Paul enjoys weight lifting, sailing, and reading.