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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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5 Best Practices For Accurate Flow Cytometry Results

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Here, we cover 5 lessons from the trenches of flow cytometry looking at important aspects of how best practices have changed over time, which practices need to be adopted, and which are outdated. Put those old, coffee-stained protocols away and take advantage of the best practices for digital instruments to write new and improved ones (coffee stains optional). Your data will thank you.

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5 Essential Calculations For Accurate Flow Cytometry Results

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Flow cytometry is a numbers game. There are percentages of a population, fluorescence intensity measurements, sample averages, data normalization, and more. Many of these common calculations are useful, but surrounded by misconceptions. This primer will help you decide which calculation to use, when to use it, and how to interpret the results.

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