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3 Compensation Mistakes That Will Ruin Your Flow Cytometry Experiments

Compensation mistakes will ruin your flow cytometry results
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Compensation is necessary due to the physics of fluorescence. Basically, compensation is the mathematical process of correcting spectral spillover from a fluorochrome into a secondary detector so that it is possible to identify single positive events in the context of a multidimensional panel. Good compensation requires that your controls tightly adhere to three rules. If the controls don’t meet this criteria, it will lead to faulty compensation resulting in false conclusions and poorly reproducible data. Even among flow cytometry veterans, a strong foundation is occasionally in need of a tune-up. And in a topic as complex as flow cytometry, it’s important that we review the fundamentals on a regular basis. In fact, it is the longtime cytometry expert who must check themselves for any sort of faith in older compensation practices. Science is ever a work in progress, and traditional methods are not always the right methods.

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7 Things You Didn’t Know About Imaging Cytometry

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It has been said that “a picture is worth a thousand words.” We are visual creatures, and we seek to capture and describe the world around us. Some of the earliest evidence for this comes from very old cave paintings found around the world, like this painting of a horse found in the caves in Lascaux, France.

With the development of reliable microscopes, such as those developed by the dutch draper Antonie van Leeuwenhoek, we were able to see what was previously invisible, probing the unseen and learning in great detail how organisms worked.

Over time, the field of cytometry (the analysis of biological processes at the whole-cell level) has expanded in many different directions. Flow cytometry can be thought of as a microscope with very poor resolution. The power of flow cytometry lies in its ability to analyze thousands of cells through many dimensions, providing an amazingly detailed understanding of the cell. However, due to the resolution, it is not possible to tell where these signals are located.

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The Truth About Flow Cytometry Measurement Compensation

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The topic of compensation is a critical one for the cytometrist to understand. It requires adherence to some specific rules, an understanding of how the instrument works, and how fluorescence occurs. Poor or incorrect compensation can easily lead to incorrect conclusions, and decreases the reliability and robustness of the data generated.
It is critical to question the wisdom of the “Protocol’s Book” and understand that the “truths” in this book are not always correct anymore. The new user doesn’t necessarily know any differently, and for this reason there are suboptimal practices that permeate flow cytometry experiments to this day.

Understanding compensation, and being armed with the knowledge, allows the researcher to combat those fairytales that continue to make their rounds in science. It is time to put them to bed and move forward with a full understanding of the process.

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Reproducibility In Flow Cytometry Requires Correct Compensation

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Understanding the 3 rules of compensation, and applying them to your everyday workflows, is an essential step in good, consistent, and reproducible flow cytometry data. Making sure the controls are bright, and treated the same way, is essential. Don’t bring unfixed controls when your samples are fixed, as the controls will not reflect the spectra from the fixed samples. Make sure not to rely on the “Universal Negative”, use a single sample to set background, and collect enough events to make sure an accurate measurement is made, as this will further improve the quality of your control and therefore the data.

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3 Requirements For Accurate Flow Cytometry Compensation

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For those new to flow cytometry, compensation is confusing at best and terrifying at worst. Likewise, those who have been doing flow cytometry since the analog ages may be holding on to practices that, while suited to the analog instruments, should be left to the annals of history. As such, a lot of time is spent discussing compensation and the best practices for this critical process. There are 3 rules that guide proper compensation, and they’ve been written about extensively since they first appeared in the “Daily Dongle” in 2011. Here, we will review the classic rules and expand upon the tacit assumptions required to fulfill them.

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