Discover The Myriad Applications Of Beads In Flow Cytometry
What is the single-most important feature of a flow cytometry experiment? Arguably, it’s the stained cells that gather data about biological processes of interest. However, a flow cytometer can measure cell-like particles as well as cells, which opens the realm of cytometry to the use of microspheres. Most researchers are familiar with the 4-Cs that beads can be used for: Control, Calibration, Compensation, and Counting. Beyond the 4-Cs, many are familiar with the multiplex bead assays for measuring analytes. Today, we will take a look beyond these well-known uses and discover the myriad applications of the “Mighty Microspheres.”
1. Receptor Quantification
As therapeutics moves into precision medicine, and with drugs being targeted to very specific receptors, it is critical that we understand the biology of these interactions. Flow cytometry is an ideal tool for these measurements. The details of these assays can be found in a previous blog, so we’ll focus on the role that beads can play in this assay. Figure 1 shows how a typical assay works. This experiment relies on three antibodies:
- The non-competing Ab, which is used to quantify the total number of target proteins on the surface of the cell.
- The target Ab, which targets the protein of interest.
- The fluorescently labeled target Ab.
The cells are first incubated with the unlabeled target Ab followed by incubation with the 2 labeled antibodies. To calculate the average receptor occupancy, a standard curve can be generated using the QuantumTM Simply CellularⓇ beads from Bangs Laboratories. These beads have different levels of antibody capture sites, allowing for quantification of the data.
Figure 1: Receptor occupancy quantification using Bangs beads.
Alternatively, beads from the BD QuantibriteTM beadset have different levels of phycoerythrin (PE) molecules, allowing for the creation of a standard curve. Because of the size of the PE molecule, the F/P ratio is 1. So, these beads make it possible to determine the number of PE molecules, which is used to calculate the antigen density.
High-speed electrostatic sorters generate thousands of droplets per second. A paper published in 2011 by Kevin Holmes showed that these droplets are small enough to both remain in the room for protracted time periods and settle in the alveoli of the lungs. The ISAC Biosafety committee has routinely recommended that cell sorter containment be tested with a variety of methods, including T4 phage, as well as different microbeads and air sampling systems. The most recent recommendations, from a paper published by Stephen Perfetto and coworkers in 2019, include the use of the Cyclex-D impact cartridge and Dragon Green beads. This new method was as sensitive as measurements performed with aerodynamic diameter measurements using an APS. Figure 2, taken from the Perfetto paper, shows the results of a test using the CyclexD cartridge.
Figure 2: The empty Cyclex D cartridge (A) and the coverslip contained within (B). The microscopic examination of the coverslip (C) shows the captured beads. The protocol recommends putting the Cyclex-D coverslip on a slide with a grid to make it easier to find the focal plane.
3. Protein-Protein Interaction Assay
Protein-protein interactions are important for understanding the biology of cells. . While several methods allow us to study these interactions, beads make an excellent base to develop such assays – especially for screening compounds that might improve or impair those interactions. Blazer and coworkers (2010) developed such an assay, which they termed the flow cytometry protein interaction assay or FCPIA.
In this assay, one protein is immobilized to a bead. The second protein is fluorescently labeled. The fluorescent signal can be monitored via flow cytometry. This system makes is possible to measure binding changes in the presence of competitors or inhibitors.
Figure 3: The FCPIA assay in brief. From Blazer and coworkers (2010).
This assay has been used in a variety of papers, including one from Storaska and coworkers (2013). In this paper, the authors looked at inhibitors of regulators of G protein signaling and screened over 300,000 compounds looking for those that would enhance a G⍺q-mediated calcium response. Typical data from this assay are shown in figure 4.
Figure 4: Typical results using the FCPIA assay. By increasing the concentration of the compound, it is possible to calculate the IC50 for compounds that inhibit RGS-G𝛼 binding. Storaska and coworkerset al (2013).
4. Detection Of Nucleic Acid Sequencings By Flow
Beads can even be used to detect nucleic acid sequences. One of these assays, BeadCon, takes advantage of something called a “black hole quencher” (BHQ). These molecules capture photons and dissipate the absorbed energy as heat. When put in close proximity to a fluorochrome, they will quench the fluorescent signal.
This assay, described in a paper by Horejsh and coworkers (2005), takes advantage of the BHQ by generating a DNA structure that has a closed conformation that places the BHQ in close proximity to a fluorochrome. When the target DNA binds to the reporter, it changes its conformation, allowing fluorescence to occur. The structures of the reporter and the assay’s functionality are shown in figure 5.
Figure 5: Left: Structure of the reporter sequence. The target DNA sequence is in the loop. The stem structure brings the fluorochrome and BHQ in close proximity while the biotin is used to bind the reporter to the microsphere. Right: The reporter is bound to a bead. When the appropriate DNA sequence binds to the loop, it causes the loop to be pulled apart, separating the fluorochrome from the BHQ, allowing fluorescence to be measured. From Horejsh and coworkers (2005).
The sensitivity of this assay was shown to be approximately 37 fmol of complementary DNA with some sequences being measured as low as 26 fmol. What makes this assay interesting and powerful is that, using a combination of beads and fluorochromes, it is possible to measure multiple DNA sequences in 1 tube. Horejsh and coworkers (2005) demonstrated this when they used two different fluorochromes and 2 bead sizes to measure 4 different respiratory virus sequences simultaneously.
Figure 6: Multiplexing beads to measure different DNA sequences. BeadCon probes for RSV and PIV-3 were generated using two different bead sizes and the reporter 6-FAM. Two different SARS genes were generated using different bead sizes and the reporter Cy5. When a complex mixture of these sequences were mixed with the beads, it was possible to measure each of the different viral sequences as shown by the black arrows. From Horejsh and coworkers (2005).
5. Protease Assays
I have a fondness for proteases, having worked on characterizing one early in my research career. I also happen to like kinetic assays in which we measure some event over time using flow cytometry. Saunders and co-workers (2010) developed a bead-based assay that filled both of these interests. In this assay, a reporter gene is constructed with a biotin tag, a putative protease cleavage site, and a fluorescent reporter (GFP). The reporter is bound to a bead and incubated with a protease of interest. If the protease cleaves the signal, it will cause the release of the reporter and thus the loss of signal on the beads. This assay can be easily used in a high throughput manner to identify protease inhibitors. It is also possible to multiplex it with different substrates and reporters. Figure 7shows how this assay works along with some representative data.
Figure 7: (Left) Schematic of the protease assay. The reporter construct consists of a biotinylation tag, a protease sequence, and a fluorescent reporter. After biotinylation, the reporter is bound to an avidin microsphere. It is incubated with the protease in question and the loss of fluorescence is measured. (Right) Typical data showing the loss of signal over time. In this case, increasing protease concentrations were added, and the loss of signal was measured. From Saunders and co-workers (2010).
The Mighty Bead may be small in size, but it possesses far-reaching potential beyond the common uses researchers put it to. In this article, we have looked beyond reliable but standard bead applications, and covered 5 different assays—from protein-binding assays and biosafety to quantitative flow and nucleic acid sequence detection. This assay shows that the versatility of these particles is limited only by the imaginations of the researchers developing and using them.
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