To make certain your instrument is set up correctly for your experiments, manufacturers have developed defined polystyrene beads. These beads’ consistent nature helps you to assess how your instrument is behaving, helps you set up proper compensation matrices, and helps you generate volumetric counts of your cell populations. Alignment, sensitivity, and fluidic quality control beads will help you to ensure that with the same wattage on the laser and the same voltage applied to the detector returns the same median fluorescence. The right compensation capture beads will bind antibodies of multiple isotypes from multiple species and give you a very…
In today’s world, many scientists have access to instruments capable of running experiments with 10 or more colors. The leap from 2 to 10 colors may seem small, but here are many factors to consider in the design and analysis of experiments that makes full use of instruments that can handle these additional colors. Imagine analyzing a 2-color experiment. With 2 biaxial plots and a single quadrant gate, you have only 4 populations to report. Now add a 3rd color. By doing so, you’ve increased your population count to 8. With 4-colors, you’ve increased your population count to 16. On…
Flow cytometrists use the Jablonski diagram to aid in understanding and explaining the kinetic events of fluorescence. Fluorescent compounds start at the ground state until they are excited by interacting with a photon of light. This photon excites the compound, promoting an electon to a higher energy state. Some of this energy is lost by emission of heat and other non-radiative processes, leading to the previous energy state. Finally, an electron falls back to the ground state while releasing a photon of light. This photon has a lower energy (higher wavelength) than the exciting photon of light. Here's how understanding…
The field of flow cytometry is moving beyond the use of isotype controls, with many suggesting they be left out of nearly all experiments. Yet, isotype controls were once considered the only negative controls you should ever use. They are still very often included by some labs, almost abandoned by others, and a subject of confusion for many beginners. What are they, why and when do I need them? Are they of any use at all, or just a waste of money? Most importantly, why do reviewers keep asking for them when they review papers containing flow data? Here is…
With the increased development of fluorescently conjugated monoclonal antibodies came more applications with potential clinical impact. In bone marrow transplantation, studies using hematopoietic cytokines made it feasible to gather stem cells from peripheral blood. It was also shown that reconstitution of bone marrow was accelerated when using cell from peripheral blood rather than bone marrow. Many more clinical flow cytoemtry applications have been developed. All of which should follow these 6 keys of running clinical flow cytometry experiments.
Flow cytometry data analysis is getting more complex. Gone is the rule of 2-3 color experiments. Even beginners are starting with 5+ color assays, and the adoption of mass cytometry has the potential to increase our headaches even more. Current data analysis methods are good for single tubes or small cohort studies. What do you do when you have a large dataset, with multiple sampling conditions, and multiple outcome measurements? With data complexity of this nature, one can export the numerical data to a third party analysis package, but even then the analysis can be difficult to perform. To overcome this…
Measuring purity is not the best way to measure flow cytometry cell sorter performance. Recovery is much more sensitive to the correct calculation of a cell sorter's drop delay than the purity. Here's how to calculate cell sorter recovery, yield, and purity, and why you should use recovery over purity to measure cell sorter performance.
What happens if one combines the power and speed of traditional flow cytometers with the resolution of a microscope? Cytometry is the study of biological processes at the whole cell level and includes techniques like light microscopy and electron microscopy. But microscopy by itself is a bit different. From the earliest days of microscopy, including the use of the first true microscopes by van Leeuwenhoek and others, scientists have been able to start seeing the finest details of a cell. With the development of the flow cytometer, researchers have been able to explore cellular processes in great deal. For example, modern…
Sudoku puzzles seem to be all the rage. I see it in coffeehouses, at the airport, even in doctors offices. Everyone is trying to work out how to fit the numbers into the grids so that everything adds up properly. Designing polychromatic flow cytometry panels is much like the Sudoku puzzle. In this case, the grid is composed of the antigens on one side, and the cytometer detectors on the other. The goal is to fill in the grid correctly. Instead of adding up to 45, like in Sudoku, the flow cytometrist is trying to optimize the ability to…
Manual compensation is the process of adjusting the compensation based on how the data visually looks. If you have manually compensated data in your lab notebook--strike it out now. Manual compensation results in overcompensated data, yielding incorrect conclusions. If you have issues, explore what those problems are and work to resolve them rather than making up fiction by manual compensation. Here are three keys to automatically compensating your data.
We all know that flow cytometry makes individual measurements on large populations of cells, it allows for statistical analysis of the data, lending strength to a researcher’s conclusions. Likewise, the isolation of very complex populations by flow cytometry cell sorting can help lead to a richer understanding of the intricate biology at the genomic, proteomic and functional level. As a reviewer of papers and grants, I am always especially interested in the details of HOW the experiments were performed because that is the critical foundation for what the data is able to tell us–and what it can NOT tell us.…
Pairing highly expressed antigens (like CD3) with dimmer fluorochromes, and the antigens of interest with the brightest fluorochromes, is a key part of panel design with few tools to help. With early generation instruments, this was relatively easy to determine, since fluorochrome choice was limited. With the advent of instruments capable of measuring more than 4 fluorochromes, there is a need to characterize the relative brightness of different fluorochromes under actual experimental conditions, rather than as free fluors. Bigos et al (2004) first reported this in an abstract and it was later simplified in Maecker et al (2004). This equation (Figure…