4 Flow Cytometry Assays For Monitoring Intracellular Processes

The most common flow assay is undoubtedly immunophenotyping, in which fluorescently tagged antibodies are used to bind to cellular proteins. This allows you to determine the types of cells present. As long as there is a fluorescent reporter available, it is possible to measure biological processes using flow cytometry – especially in a phenotypically defined manner. Probably the most common of these assays is the calcium flux assay. And that is just the tip of the iceberg.

In addition to calcium, it is possible to measure magnesium and zinc concentrations, reactive oxygen species, and even membrane potential using flow. Today, we’ll cover 4 assays that use a fluorescent reporter to measure their target, allowing researchers to challenge the cells and measure their response in real time.

1. Calcium Flux

Secondary messengers play an important role in cellular homeostasis. By responding to the first messengers, these compounds trigger a variety of cellular processes ranging from proliferation to apoptosis, differentiation, and more. One such secondary messenger is Calcium (Ca²⁺). Calcium levels in both the blood and cells are tightly regulated. This is because calcium is such a potent initiator of biological processes.

Several compounds can fluoresce in the presence (and absence) of calcium, and these are shown in Table 1.

Table 1: Common Ca²⁺ indicators

The first two dyes allow for ratiometric measurements, which means that we can track the ratio of bound-to-free calcium, making the assay resistant to differences in loading conditions between experiments. However, Indo-1 requires a UV laser, while Fura-red requires a violet laser. Measurements made with a single color have to be more tightly controlled for loading.

The general design of a Ca²⁺ flux experiment is such that cells are loaded with the appropriate dye and kept at room temperature.

The power of flow to probe biological processes, at the whole-cell level and in a phenotypically defined manner, remains unchallenged. Tubes are loaded onto the flow cytometer, and a baseline of 30-to-60 seconds is run before the agonist is quickly added to the tube.

The flux is monitored by the change in fluorescence of the reporter. After the signal has stabilized for a period of time, an ionophore, such as ionomycin is added to establish the maximal possible calcium flux. A typical data analysis is shown in Figure 1.

Figure 1: Typical Ca²⁺ data analysis. T-cells were labeled with indo-1 and incubated with fluorescently labeled antigen-presenting cells (APC) that were either wild-type (633 conjugate, red line) or mutants that could not easily form conjugates (488 conjugate, blue line). After an initial baseline, the cells were forced to form conjugates, and changes in Indo-1 fluorescence were measured. After about 10 minutes, the cells were treated with ionomycin to confirm that the T-cells were responsive and the maximal calcium response was established. Data from Graf et al., 2007.

One limitation of these assays is that it is almost impossible to catch the instantaneous flux when the agonist is added. With the release of the Accuri, this became less of an issue. Vines et al., (2010) demonstrated that using the Accuri, it was possible to add the agonist while still acquiring the data. The disadvantage was that it required the use of Fluo-4, a non-ratiometric dye. The data are shown in Figure 2.

Figure 2: Comparison of Ca2+ measurements using a Cyan, which requires the tube to be removed to add agonist, and the Accuri, which does not. On the right is a photograph of a researcher adding agonists to a tube on the Accuri. From Vines et al., (2010).

This assay can easily be performed to look at phenotypically defined cells, as was shown in this paper by Quách et al., (2011). Here, the authors examined the ability of an IgM-expressing cell’s ability to flux Ca2+ after stimulation with either anti-IgM or anti-IgD. Results from this paper are shown in Figure 3.

Figure 3: (A) identification of naive B cells. (B and C) Ability of cells to flux calcium (top) and the percent of responding cells (bottom) after either anti-IgM (B) or anti-IgD(C). Details can be found in Figure 1 of Quách et al., (2011).

Clearly, the calcium flux assay is a powerful tool for measuring changes in ion concentration in phenotypically defined cells, and with the proliferation of UV lasers in newer instruments, this assay is due for a resurgence in popularity.

2. Reactive Oxygen Species (ROS)

Reactive oxygen species (ROS) also act as secondary messengers in cells. Depending on the concentration, ROS can be good or bad for the cell. On the plus side, ROS are involved in cell signaling, homeostasis, and apoptosis. For example:

• NF𝜅B activation may require ROS
• IL-1 induces insulin-like growth factor binding protein-1 synthesis via ROS mechanism
• IL-1β requires ROS produced by 5-LOX to activate NF𝜅B
• Platelets release ROS at the site of injury, which recruits more platelets
• TNF-⍺ activation of ASK-1 requires ROS

On the downside, ROS can cause damage to cells in a variety of ways, including:

• Damage of DNA or RNA
• Oxidations of polyunsaturated fatty acids in lipids
• Oxidations of amino acids in proteins
• Oxidative deactivation of specific enzymes by oxidation of co-factors
• Cancer cells have higher ROS levels than normal cells
• ROS may play a role in memory decline with age

There are several dyes that can be used for measuring different ROS species, and these are listed in Table 2.

Table 2: Different dyes for ROS detection. From Thermofisher:

ROS measurements can be used to diagnose conditions, such as Chronic Granulomatous Disease (CGD). This hereditary disease is marked by the inability of cells to make ROS due to a defect in phagocyte NADPH oxidase. An example of this assay was illustrated in this ICCS newsletter, shown in Figure 4.

Figure 4. Cells are labeled with DHR, and a baseline ROS measurement is made (top row). Upon stimulation with PMA, normal cells produce ROS, and the ratio of stimulated to unstimulated cells can be calculated (NOI). As shown in the lower middle, the patient’s cells do not produce ROS upon stimulation (indicated by the NOI of 1). Interestingly, the patient’s mother’s cells showed two peaks: a normal one (NOI of 830) and one with a compromised ROS production (NOI of 21). This illustrated that it was a case of p47Phox CGD.

3. Zinc

Zinc is a required trace element that is used in over 300 proteins implied in processes like nucleic acid metabolism, cell replication, and tissue repair. Deficiencies in zinc can lead to a variety of issues ranging from impaired immunity to stunted neuronal development and cancer. The cell-permeable reporter Zinpyr-1 has a maximum excitation of 515 nm and a maximum emission of 525 nm, which makes it suitable for flow cytometry applications. Malavolta et al., (2006) used this probe to estimate the availability of zinc in CD4 and CD8 T-cells. Figure 5 shows the results of this work.

Figure 5: (A) untreated (B) treated with a NO donor (C) treated with the zinc chelator TPEN. The panels on the left show histograms of the Zinper-1 fluorescence after treatment. Data from Malavolta et al., (2006).

4. Magnesium

Magnesium is an important enzyme cofactor in biological processes, including energy production, oxidative phosphorylation, and ion transport. Deficiency can cause increased cardiovascular risk and insulin resistance. In a study by Fox et al., (2007), the use of a fluorescent probe, Magnesium Green, helped the researchers to calculate the intracellular magnesium in platelets.

The logic behind this is that platelets are easy to isolate and can provide a measurement of intracellular magnesium.

To generate a standard curve, the team incubated platelets in solutions with different concentrations of magnesium. Ionomycin was added, and the Mean Fluorescence Intensity (MFI) was measured. Additionally, platelets were treated with ionomycin in the absence of external magnesium. An MFI of the sample was measured in the absence of ionomycin. Typical data are shown in Figure 6.

Figure 6: The standard curve was generated as described above, and the best-fit curve was calculated. Using the MFI of the test sample, it is possible to calculate the intracellular magnesium concentration. Data from Figure 2 of Fox et al., (2007).

This method allows for more rapid calculation of the intracellular magnesium, which is a better measure than serum magnesium – again, showing the power of flow cytometry.

In conclusion, we’ve just covered 4 assays that use fluorescent reporters to measure their target. Calcium flux, ROS, zinc, and magnesium. These assays allow researchers to challenge the cells and measure responses in real time, demonstrating the power of flow cytometry as a tool for measuring changes in intracellular ion concentrations.

As long as you have access to a fluorescent reporter, you can measure biological processes using flow cytometry. This can be done in combination with phenotyping to understand the ions being perturbed in specific cellular subsets. Many other ions can be measured, we will discuss these in a future blog.

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