Flow cytometry is used to measure the physical and biochemical properties of cells and cell-like particles using fluorescence.
It is crucial that each individual particle suspension (within a defined size range) can pass through the flow cytometer.
For better or for worse, beads are a must-have for the flow cytometrist. From quality control to standardization to compensation, there is an account for every job. They are important, even critical, to flow cytometry.
Beads can do a lot to improve flow cytometry, so without further ado, let's dive into the world of beads.
1. Quality Control Accounts.
Quality control accounts start at the top. These beads represent the first line of defense for any facility to determine if their instrument is performing within specifications set by the vendor or, more importantly, the facility itself.
Over time, the peculiarities and quirks of each instrument are revealed as quality controls are performed and data analyzed.
It is important to ensure that you analyze trends in the QC granule so problems can be identified before they become an actual problem.
Before the days of automated quality control protocols, every facility had to do thisSet up your own quality control program. My favorite bead set back then was the AlignFlow series from Molecular Probes (now ThermoFisher). In this series, a different bead had to be optimized for each laser. Fortunately, there are now better options.
In order to automate the quality control of the BD series digital cytometers, Cytometry Setup and Tracking (CS&T) was introduced at DIVA in the mid-2000s. This protocol and associated beads provided an automated way to collect quality control data and most importantly, track it.
This was of great help to central facilities trying to maintain instrument quality control. However, it was limited to BD instruments, and if the instrument was one of their Special Order Search Products (SORPs) or had a non-standard filter arrangement, CS&T could have problems.
illustration 1: Quality control reports using the CS&T system.
Cytek recently released another automated quality control system that can be used on many flow cytometers. Your program and associated accounts are referred to as the QbSure system.
In this case, the software takes measurements of Q (detector efficiency) and b (background noise) as well as an additional metric called R.
The value of R helps characterize a system's resolution, and a smaller R is better.
Figure 2, taken from the Cytek website, shows the R values for 3 different instruments and the resulting scatterplot for a CY7-PE stained sample with low expression levels. Notice how the difference between negative and positive increases as the R value decreases.
Figure 2: The effect of the R value on detecting a weak signal.Make site da Cytek.
2. Compensation particle
Let's face it, compensation is a necessary pain. Followingthe 3 remuneration rulesand using automated algorithms are best practices for this process.
The support that accompanies the fluorochrome to the cut point is not important as long as the positive and negative populations exhibit the same autofluorescence.
So convenience matters. If you have plenty of cells, say from a spleen, and all antigens are adequately expressed, cells are fine to use. However, if you do not have extra cells, have weak antigens, or are targeting rare cells, using the sample cells as a control is less attractive.
Insert the antibody capture bead. Sold by many vendors under different names, these are plastic pellets coated with an antibody that can bind to part of another antibody.
It can be the light chain or the heavy chain, or it can be species specific. you can even useProtein-AÖG-ProteinAntibody granules that will not work with other granules.
Closing accounts has several advantages. First, the beads bind to any antibodies in the solution, resulting in a bright, low CV signal. Compare the plots in Figure 3 of cells labeled FITC (left) or beads (right).
It is much easier to positively identify the positive population with the counts. In addition, this ensures that the first compensation rule is satisfied.
Figure 3: Comparison of granules vs. cells. Cells (left) or antibody capture beads (right) were stained with the same FITC-conjugated antibody. The middle graphic shows the overlap of cells and accounts. The black dashed line shows the lower limit of the positive cable signal. These plots show that it is easy to separate the positive from the negative and that the positive signal is at least as bright as the experimental sample.
Obviously, the second rule is easy to follow as long as a positive and negative count is identified in each control.
Avoid the urge to use a universal negative or, even worse, cells as the negative.
As shown in Figure 4, if unstained cells were used as the negative population, the compensation would be wrong because the cells have a different background fluorescence than the beads.
Figure 4: Identifying the appropriate negative population is critical for accurate compensation.
Finally, the use of beads ensures that the controls obey the third and final compensation rule: namely, that the compensation control and the experimental sample must be collected under identical conditions. That means the same treatment (e.g. fixed or unfixed), with the same antibody with the same sensitivity, don't touch that voltage!
3. Count bills
If an absolute cell count is required, there are a few options. First, you have access to a volumetric system (MACSQuant, Accuri, Attune, etc.) that has a very accurate measurement of the volume of sample injected into the system.
That being said, the next best thing is a counting account. These are accounts that have a very precise known count, so it's possible to calculate the cells using a ratio.
They are available in 2 different preparations. The first preparation is a tube containing a precise number of balls. A defined sample volume is placed in the tube. The sample is processed in the flow cytometer and the absolute number of samples is:
The second bead preparation is already in solution and the examiner places a defined amount of beads in the tube of interest. If the number of beads added is known, a similar calculation can be performed to determine the cell concentration in the tube.
It is important to note that in both cases, accurate pipetting is critical.
4. GloGerm-Content (other YG-Content)
The biosecurity of cell sorters is an important issue for sorting operators. ISAC has published guidelines forBiosafety Classifier, it's himNIH campusalso adopted specific rules.
If your facility does not have rules, now is a good time to discuss these best practices with them and your biosafety office.
An important part of biocontainment validation is testing to ensure engineering controls are working correctly.
This can be done with a cell replacement (a shiny bead) and an air sampler.
The initial work was done with a particle called the "GloGerm Bead" (and yes, you can buy it on Amazon.com). Highly fluorescent under a black light, these spheres are great for teaching hand washing, aseptic technique, and spreading disease through physical contact.
Early work characterizing the efficiency of engineered controls used these spheres. Unfortunately, they had 2 disadvantages:
- Having to be extensively washed out of solution, these grains were brought in to bring them into an aqueous solution.
- The bullets were of mixed sizes and could stick to dust particles.
Enter Polyscience Yellow-Green (YG) Beads, which are highly fluorescent uniform particles available from 0.5 µm to 10 µm.
This gem and data collection system was first discussed at the ISAC XXII International Conference by Hank Pletcher and Jonni Moore of the University of Pennsylvaniaenvironmental monitoring systemsoffer a cost-effective and easy-to-use system for monitoring the biocontainment of a cell sorter.
The heart of this system is theCyclex-DCartridge, which is a sealed container connected (via a hose) to a pump. When air enters the Cyclex-D cartridge, it flows over the sticky coverslip and any airborne particles stick to the coverslip.
After sampling, the cartridge is opened and the coverslip is examined under a microscope for the presence of grains.
Figure 5: YG test results of a FACSAria. (A) The test did not show that the Cyclex-D cassette worked. (B) No errors with AMS enabled. (C and D) Two independent 10' error runs with AMS on, no beads were detected on either coverslip.
The containment test draws 200 liters of air (at a rate of 20 liters/minute). Because the Cyclex-D cartridge is small, it can be placed at any distance from where you want to sample.
The Cyclex-D system is a wonderful system for containment validation and is highly recommended for all cell screening facilities.
5. Standardization Accounts
This is a broad category of beads that can be used for a variety of different techniques. One of the most common is determining the number of antibodies bound to the cell.
If PE-labeled antibodies are used, well-characterized beads can be used since the F/P ratio for PE is typically 1:1 due to steric constraints.
If the antibody is labeled with another reagent, thatsimply quantum cellPearls are a great option.
This bead set consists of 5 beads, 1 unstained and 4 with an increasing number of antibody binding sites. The beads are labeled and processed in a flow cytometer, allowing a standard curve to be generated as shown below:
Figure 6: Standard curve generated with Simply Cellular Beads to determine antibody binding capacity (AUC) number. The dashed lines represent the 95% confidence level.
Using a regression equation it is possible to take the MFI of the unknown sample and calculate the AUC. It is thus possible to determine the binding sites of antibodies in a target cell.
This technique can be useful in experiments such asreceiver cast. Another way to use this technique is to standardize the AUC to a target of interest across multiple experiments.
Microspheres are a very useful item in the flow cytometrist's tool kit. Each type of bead has a different use to improve understanding of how the instrument or experiment works, and care must be taken not to overinterpret the bead's results. Using beads makes troubleshooting experiments and instrument problems a breeze.
When the PI growls
When paper is rejected
If Fortessa falls... again
I only remember my favorite accounts
And so I experience it again
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