5 Essential Beads For Flow Cytometry Experiments

Flow cytometry is designed to measure physical and biochemical characteristics of cells and cell-like particles using fluorescence.

Fundamentally, any single-particle suspension (within a defined size range) can pass through the flow cytometer.

Beads, for better or worse, are a sine qua non for the flow cytometrist. From quality control, to standardization, to compensation, there is a bead for every job. They are important — critical, even — for flow cytometry.

Beads can do much to enhance flow cytometry, so without further ado, let’s delve into the world of beads.

1. Quality Control Beads

Starting at the top are the quality control beads. These beads represent the first line of defense for any facility to determine if their instrument is working within the specifications set by either the vendor or, more importantly, the facility itself.

Over time, each instrument’s quirks and idiosyncrasies are revealed as quality control beads are run and the data analyzed.

It is critical to make sure to analyze the trends in QC beads so that issues can be spotted before they become a real problem.

Before the days of automated QC protocols, each facility had to establish their own QC program. My go-to beadset at the time was the AlignFlow series from Molecular Probes (now ThermoFisher). This series required a different bead for optimization of each laser. Luckily, there are better options now.

In an effort to automate QC on the BD line of digital cytometers, Cytometry Setup and Tracking (CS&T) was introduced to DIVA in the mid-2000s. This protocol, and the associated beads, provided an automatic way to collect QC data and, more importantly, track it.

This was a huge boon to the core facilities trying to keep on top of instrument QC. However, it was limited to the BD instruments, and if the instrument was one of their Special Order Research Products (SORP), or had a non-standard filter arrangement, CS&T could have difficulties.

Figure 1: QC reports using the CS&T system.

Recently, another automated QC system that can be used on many flow cytometers was released by Cytek. Their program, and associated beads, is called the QbSure system.

In this case, the software makes measurements of both Q (detector efficiency) and b (background noise), as well as an additional metric called R.

The R value helps to characterize the resolution of a system, and a lower R is better.

Figure 2, taken from the Cytek website, shows R values for 3 different instruments and the resulting scatter plot for a CY7-PE stained sample with low levels of expression. Notice how, as the R value decreases, the separation between negative and positive increases.

Figure 2: The effect of R value on detection of a dim signal. From Cytek website.

2. Compensation Particles

Let’s face it, compensation is a necessary pain. Following the 3 rules of compensation and using automated algorithms is best practices when it comes to this process.

The carrier that escorts the fluorochrome to the intercept is not important, as long as the positive and negative populations have the same autofluorescence.

So, it comes down to convenience. If you’ve got abundant cells, say from a spleen, and the antigens are all reasonably expressed, using cells is fine. However, in the case where you don’t have extra cells, you have dim antigens, or are looking for targets on rare cells, using the sample cells for a control is less appealing.

Enter the antibody capture bead. Sold by many vendors under different names, these are plastic beads that are coated with an antibody that can bind some part of another antibody.

It could be the light chain or the heavy chain, or it could be species-specific. You can even use Protein-A or Protein-G beads for antibodies that don’t work with other beads.

There are several advantages of compensation beads. First, the beads bind all the antibody in the solution, resulting in a bright signal with low CV. Compare the plots in Figure 3 of FITC labeled cells (left) or beads (right).

It is much easier to cleanly identify the positive population with the beads. Additionally, this ensures that the first rule of compensation is met.

Figure 3: Comparison of beads vs. cells. Cells (left) or antibody capture beads (right) were stained with the same FITC conjugated antibody. The center plot shows the overlay of the cells and beads. The black dashed line shows the lower limit of the bead positive signal. It is clear from these plots that it is easy to gate the positive from the negative, and that the positive signal is at least as bright as the experimental sample will be.

Of course, the second rule is easy to meet, as long as one identifies a positive and negative bead in each control.

Avoid the desire to use a universal negative or, worse yet, cells as a negative.

As shown in Figure 4, if unstained cells were used as the negative population, the compensation would be incorrect, as the cells have a different background fluorescence than the beads.

Figure 4: Identification of the proper negative population is critical for accurate compensation.

Finally, using beads ensures that the controls meet the third and final rule of compensation: that is, that the compensation control and experimental sample must be collected under identical conditions. This means the same treatment (e.g. fixed or unfixed), with the same antibody at the same sensitivity — don’t touch that voltage!

3. Counting Beads

If an absolute count of cells is needed, there are a few options. First, is to have access to a volumetric driven system (MACSQuant, Accuri, Attune, etc.), which has a very accurate measure of the volume of the sample that is injected into the system.

Barring that, the next best thing is a counting bead. These are beads that have a very precise, known count so that it is possible to calculate cells using a proportion.

They come in 2 different preparations. The first preparation is a tube containing a precise number of beads. A defined volume of sample is added to the tube. The sample is run on the flow cytometer and the absolute count of the sample is:

The second preparation of beads comes already in solution and the researcher adds a defined amount of beads to the tube of interest. Knowing the amount of beads added, one can do a similar calculation to determine the concentration of cells in the tube.

It is important to note that in both of these cases, accurate pipetting is critical.

4. GloGerm Beads (or YG beads)

Biosafety of cell sorters is an important issue for sort operators. ISAC has released guidelines for sorter biosafety, and the NIH campus has adopted specific rules as well.

If your facility doesn’t have any rules in place, it is a good time to talk to them — and your Biosafety office — about these best practices.

One important component of biocontainment validation is a test to ensure that the engineering controls are working correctly.

This can be done using a surrogate for cells (a bright bead) and an air sampler.

The initial work was done using a particle called the “GloGerm bead” (and yes, you can buy them on Amazon.com). These beads are highly fluorescent under blacklight, and are great to teach about hand washing, aseptic technique, and the spread of disease by physical contact.

Early work in characterizing the efficiency of engineering controls used these beads. Unfortunately, they had 2 downsides:

1. They needed to be washed extensively from the solution, these beads came in to get them into an aqueous solution.
2. The beads had mixed sizes and could bind to dust particles.

Enter the Polyscience Yellow-Green (YG) bead, which are a highly fluorescent and uniform particle available from 0.5 µm to 10 µm.

First discussed at the ISAC XXII International Conference, by Hank Pletcher and Jonni Moore from the University of Pennsylvania, this bead and a collection system from Environmental Monitoring Systems offer an inexpensive and easy-to-use system for monitoring biocontainment of a cell sorter.

At the heart of this system is the Cyclex-D cartridge, which is a sealed container that attaches to a pump (via tubing). As air is drawn into the Cyclex-D cartridge, it passes over sticky coverslip, and any particles in the air stick to this coverslip.

After sampling, the cartridge is opened and the coverslip inspected under the microscope for the presence of any beads.

Figure 5: Results of YG testing of a FACSAria. (A) Test Failure to demonstrate Cyclex-D cassette worked. (B) No Failure with AMS on. (C and D) Two independent tests of a 10’ failure with the AMS on, no beads were detected on either coverslip.

In testing the containment, 200 liters of air (at a rate of 20 liters/minute) is sampled. Since the Cyclex-D cartridge is small, it can be placed at any distance from the point you wish to sample.

The Cyclex-D system is a wonderful system to use for containment validation, and strongly recommended for all cell-sorting facilities.

5. Standardization beads

This is a broad category of beads that can be used for a variety of different techniques. One of the most common is the determination of the number of antibodies bound to the cell.

If using PE labeled antibodies, well-characterized beads can be used, since the F/P ratio for PE is generally 1:1, due to steric constraints.

If the antibody is labeled with another reagent, the Quantum Simply Cellular beads are a great option.

This beadset has 5 beads, 1 unstained and 4 with increasing numbers of antibody binding sites. The beads are labeled and run on a flow cytometer allowing for the generation of a standard curve, as shown below:

Figure 6: Standard curve generated using the Simply Cellular beads to determine the number of antibody binding capacity (ABC). The dashed lines represent the 95% confidence level.

Using a regression equation, it is possible to use the MFI of the unknown sample, and calculate the ABC. Thus, it is possible to determine the antibody binding sites on a target cell.

This technique can be useful in experiments like receptor occupancy. Another way to use this technique is to standardize the ABCs on a target of interest across multiple experiments.

Beads are a very useful item in the flow cytometrist’s toolkit. Each class of bead has a different use for improving the understanding of how the instrument or the experiment is performing, and care must be taken not to over-interpret the bead results. By using beads, troubleshooting experiments and instrument issues becomes a breeze.

When the PI growls

When the paper’s rejected

When the Fortessa is down… again

I simply remember my favorite beads

And then I’m experimenting again

To learn more about the 5 Essential Beads For Flow Cytometry Experiments, and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on the Flow Cytometry Mastery Class wait list.