How To Achieve Accurate Flow Cytometry Calcium Flux Measurements

Most flow cytometry experiments work with antibodies conjugated to a fluorochrome for some variation on immunophenotyping. However, any fluorochrome that is excited by one of the available excitation sources, and emits within the range of the detectors, can be incorporated into an experiment.

One of the great pleasures of the past was leafing through the Molecular Probes handbook, seeing what fluorescent dyes had just been released, and thinking of possible applications for them. The classic example of non-antibody directed fluorochromes are DNA-binding dyes like PI, 7-AAD, and Hoechst, but there are many others.

Dyes exist for the detection of everything from large nucleic acids to reactive oxygen species, and from lipid aggregates to small ions. Concentrations of physiologically important ions such as sodium, potassium, and calcium can be important indicators of health and disease.

Calcium ions play an especially critical role in cellular signaling. As a signaling messenger, calcium is involved in everything from muscle contractions, to cell motility, to enzyme activity.

Cells tightly regulate calcium to ensure that the cytoplasmic concentration of Ca²⁺ is in the 100 nM range.

Mechanisms of calcium homeostasis include sequestering Ca²⁺ in different organelles and proteins, as well as actively secreting excess calcium from the cell.

Clearly, a probe to monitor calcium dynamics in real time would be valuable.

Fortunately, there are several different fluorochromes available for measuring changes in calcium levels (flux) by flow cytometry. A complete list can be found here.

The first Ca²⁺-responsive fluorochromes were published by Tsien in 1980 and, as described in the Molecular Probes Handbook, were later improved by Haugland.

Modern flow cytometrists have 2 classes of dyes available to them: those that respond to an increase in calcium, and those that respond differently to both free calcium and a lack of calcium (ratiometric).

When preparing for a calcium flux experiment, there are a couple of things that need to be considered for designing the experiment.

1. What excitation sources are available for use? Most of the common calcium-sensing fluorochromes are excited off a 355 nm, 405 nm, or 488 nm laser. If the plan is to couple the calcium flux assay to immunophenotyping in an effort to see which cells are responding to a given stimulus, choosing a calcium-sensing dye is an important decision.With the increased presence of UV lasers on systems (due in part to the brilliant UV dyes), the use of UV-excitable Indo-1 is a great choice, allowing for phenotyping off of other lasers.
2. Sensing or Ratiometric Calcium dye? The advantage of using a ratiometric dye — a dye whose excitation or emission peak changes in the presence of calcium — is strongly recommended. This ensures that the dye loading step of the assay is less critical than for the dyes that only respond to the presence of calcium.
3. Temperature control? How critical is it to have biologically relevant temperatures for the response? Although cells will flux at room temperature, the kinetics are different at 37 ℃. There are several ways to achieve temperature control for flow samples. Necessity is the mother of invention, and using tools common to fans of the TV show MacGyver, it is pretty easy to create your own temperature control system using an aquarium submersible pump, an old water bath, some plastic tubing, and waterproof tape.Take a 5 ml tube that fits on your flow cytometer, wrap 4-6 coils around it, and secure them with tape very tightly. The goal is to make a jacket for the tube.Next, connect the tubing to the pump, and place in the water bath. The final step is to calibrate the system: put your sample solution in the tube, and add a thermometer. Set the water bath to a couple of degrees above desired temperature in the tube, and monitor it.Make sure you have a stack of paper towels or absorbent benchpads handy, as leaks could spring at any moment!
4. Capturing initial Ca²⁺ response? On most flow cytometers, you have to take the sample tube off to add the stimulus. This means that the earliest Ca²⁺ response is missed. That is why there is usually a break in the data right after the unstimulated baseline.With the advent of more syringe- and peristaltic pump-based systems, this may become a moot issue for future experiments.

Calcium Responsive Probes

The first class of Ca²⁺ probes are those that increase fluorescence in the presence of free calcium.

Two of the prototypical dyes are shown below, in Figure 1. Fluo-3 and Fluo-4 are both excited by 488 nm light, and fluoresce in the low 500 nm range. Based on data from the Molecular Probes Handbook, Fluo-4 has a better response, compared to Fluo-3, in a FLIPR readout. The original paper describing Fluo-4 also indicated that it performed better than Fluo-3. As with everything in flow cytometry, make sure to test the reagents that will be used in the assay.

Figure 1: Fluorescence spectra of Fluo-3 and Fluo-4.

There are several additional probes that can be used in this mode.

To use these, cells are first loaded with a membrane-permeant version of the dye, which is then cleaved by esterases to release a charged version that remains trapped in the cell.

Dye titration should be performed to optimize loading conditions for each cell type.

For difficult-to-load samples, addition of 0.01-0.02% Pluronic acid has been shown to facilitate dye loading. It is critical to keep cell concentrations consistent from run to run.

Single response dyes are easy to use and require only lasers that are available on pretty much every instrument in the field. Unfortunately, different levels of loading will often result in different responses.

The second class of Ca²⁺ probes are the ratiometric dyes.

The most commonly used ratiometric dye is Indo-1. It is excited by the UV laser and shifts its emission spectrum when it is bound to calcium (Figure 2). By evaluating the ratio of free to bound, a more accurate and loading-independent kinetic reaction can be measured.

Figure 2 : Excitation and Emission profile for Indo-1.

For those instruments that don’t have a UV laser, it is possible to Fura-Red as a ratiometric dye. In the absence of Ca++, the dye would be best excited by the 488 nm laser. In the presence of Ca++ the best excitation would be off the 405 nm laser (Figure 3). Thus, the ratio of the emission off the 405 nm laser divided by the emission off the 488 nm laser will provide a ratiometric response similar to Indo-1.

Figure 3: Excitation and Emission profile for Fura-Red

Running a Calcium Flux Experiment

Gather the cells and label with the dye of choice. Keep the unused cells in the dark at RT while conducting a flux. Make sure that the stimulation reagents are ready and that a pipet is dialed in to deliver the correct amount of stimulant to the cells.

Another reagent to have on hand is a calcium ionophore. The 2 most common compounds used are ionomycin and A23187.

When added to cells, the ionophore will shuttle calcium ions across the plasma membrane to cause the maximal response in the cells.

While some protocols will have the ionophore control run on a separate tube, it is often better to add ionomycin at the end of the acquisition to get a measurement of the maximal fluorescence of the cells. You then have data for baseline, stimulated, and maximal calcium response for each tube.

With early instruments for ratiometric measurements, it was necessary to collect the data, trying to balance the 2 emissions. With newer instruments, a ratio can be set up and collected at the time of data acquisition.

This is achieved by making sure the signal from both dyes is on-scale and, having a histogram plot — or better yet, a plot of ratio vs time — and making sure that the ratio of the dyes is in a reasonable place (somewhere along 1000) as shown in Figure 4. And yes, you do run calcium flux in linear scale.

A typical analysis is shown in Figure 4 from Graf et al. (2007). In this experiment, the authors used Indo-1 to examine calcium flux in T-cells that had formed conjugates with wild type (red) or mutant (blue) antigen-presenting cells.

There is an initial baseline to establish the fluorescence level. The first arrow indicates where the conjugates were formed, which causes a break in the data.

The red line shows an increase in calcium flux, indicating that the wild-type APCs induce a calcium flux in the T-cells. The blue line doesn’t change, demonstrating that the mutant APCs are not capable of inducing a calcium flux. The green line, representing unbound T-cells, do not flux calcium either.

At the second arrow, a little over 10 minutes from the start of the experiment, the authors added Ionomycin. This causes an influx of calcium into the cells and, as can be seen, all of the T-cells showed a positive calcium response.

Figure 4: Figure 6c from Graf et al. (2007), showing the typical analysis of a calcium flux experiment.

Notice how the authors cleaned up the data by plotting the median fluorescent intensity at each time point. Additionally, the data is smoothed, which helps reduce noise. There are several common methods for smoothing data.

The first method is a moving average. In this process, at time point t, the mean of n to n+25 is calculated. At t+1, the average of the next N data points (n+1 to n+26) is calculated, and so on. The moving average value can be chosen based on the experimental needs.

A second method is to use a Gaussian smoothing, which places less weight on values farther away from the center. The choice is up to the investigator.

This graph shows the one limitation on traditional analysis, and that is the break that prevents the detection of the earliest calcium flux. There used to be a system, called the Time Zero System, sold by Cytek that was able to get better measurements of the initial calcium flux.

With the advent of newer syringe-driven systems, there became another option, as demonstrated in Vines et al (2010).

Using the Accuri C6, which has a pump system, the sample tube is not under pressure so it is possible to add stimuli directly while continuing to acquire data. Figure 5 below, taken from Figure 2 from the Vines paper, shows how this data looks on the Accuri and a Cyan.

Figure 5: Figure 2 from Vines et al (2010), showing how having the ability to add stimuli without removing the tube from the sip allows for the earliest calcium flux to be captured.

In this paper, the authors were limited to using Fluo-4, so the data could not be acquired in a ratiometric manner. With the improvements on the newer cytometers, it should be possible to perform similar experiments and capture this early response using a ratiometric measurement.

To summarize:

• Start with identifying the instrument to be used, which will dictate what fluorochromes can be used.
• Use a ratiometric dye whenever possible, because this allows the data to be acquired without concerns over dye loading differences.
• Make to sure have a reagent, like ionomycin, to determine the maximal fluorescent signal.
• Choose an appropriate smoothing model.
• Decide how best to present data (graphically, fold over control, etc.).

Calcium experiments can be very informative and, with the advent of cheaper UV lasers, more and more researchers can use ratiometric measurements to evaluate the signaling processes in phenotypically defined populations.

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