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The Truth About Flow Cytometry Measurement Compensation

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In most research labs, there exists a notebook that contains the tried and true protocols for lab members to follow. These hallowed, often coffee-stained, pages teach the researchers everything — from how to make media, passage cells, and run restriction digestions, to how to prepare cells for flow cytometry analysis. These protocols are time-honored and tested, so the new researcher doesn’t question the wisdom of the “Protocols Book”.

Unfortunately, these pages are not refreshed with the best practices that have evolved over time as the technology and our understanding has changed and grown. The “truths” in this book are not always right anymore, but the new user doesn’t necessarily know any differently. It is for this reason that there are suboptimal practices that permeate flow cytometry experiments to this day. The last 2 blog articles have discussed the theory and practice of compensation. This blog article will help shine light on some of these historical practices and why they need to be changed.

You can use a universal negative

The idea behind the Universal Ne ...

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Reproducibility In Flow Cytometry Requires Correct Compensation

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Why do we have to compensate flow cytometry data?

Newcomers to flow cytometry are often confronted with one of the most confounding issues in flow cytometry. That is, trying to understand the whole idea of “compensation”. It can be explained theoretically, mathematically, by trial and error, or by “take my word for it”. Depending on the audience, a combination of these are used to get the point across.

Simply put, compensation is the mathematical process of correcting the spectral spillover of a fluorochrome into a secondary detector. It relates to the physics of fluorescence. To understand what this means, let’s start with the Jablonski diagram of fluorescence.

Figure 1: Jablonski diagram of fluorescence. Used user creative commons license. Original.

A fluorescent molecule starts at rest, with electrons in the ground state. When a photon of light hits this molecule, it is absorbed (purple line), promoting an electron to a higher energy state. There are a variety of ways that the energy release can happen — we are specifically interested in fluorescence. When a mole ...

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Best Practices In Flow Cytometry Compensation Methodologies

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What is compensation and when should you do it?

In this first of 3 blog articles, we will discuss the principles of compensation, as well as why it is important, and how to perform compensation. Subsequent articles will discuss the rules that must be followed for proper compensation and some of the common compensation myths that permeate the field. It all begins with an understanding of the process of fluorescence.

After excitation, a fluorescent molecule emits a photon. This photon has an emission maximum — that is, the most probable photon wavelength that will be emitted. However, this emission is not so specific, and there are a range of photons that can be released from the molecule. This can be modeled with a variety of software. A typical emission profile for a common fluorochrome, fluorescein, is shown in Figure 1.

Figure 1: Fluorescein emission profile.

As can be seen from this spectra, Fluorescein has a maximal emission of about 524 nm. However, it has a very long tail, and there is a chance (albeit small) that a photon of over 600 nm can be emitted by this molecule. Flu ...

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The Need For Speed In Flow Cytometry Data Analysis

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Speed is a highly touted metric in flow cytometry. Look at any vendor’s website and you will see the highlights on how many events per second their instrument can acquire, how many cells can be sorted per second, and more. The limitations are imposed by the physics of flow cytometry, the speed of pulse processing, and more. With cell sorters, Poisson statistics dominate the speed calculation. As has been discussed before, the optimal sort rate is ¼ the frequency of droplet generation. Sorting faster will impact purity of the final product.

One of the trends in flow cytometry is pushing the limit of the number of parameters that can be measured at one time. The CyTOF threw the gauntlet down to start this new race by changing how the signal was detected. It didn’t take long for fluorescence-based cytometers to begin pushing past the 18-fluorochrome limit, and now instruments that can do 24 or more fluorescent parameters at the same time are available. Spectral cytometry may push this limit to 50 parameters or more in the near future.

With all these parameters, the data files bec ...

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Instrument Quality Control For Reproducible Flow Cytometry Experiments

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The NIH has released a series of reproducibility guidelines that scientists must address. These guidelines have been introduced because there’s a lot of data showing that reproducibility in science is frustratingly lacking in certain experiments.

Non-reproducible experiments waste time, money, and resources. As Begley and Ioannidis cite in their 2015 article :

“The estimates for irreproducibility based on these empirical observations range from 75% to 90%. These estimates fit remarkably well with estimates of 85% for the proportion of biomedical research that is wasted at-large.”

Reproducibility is a mindset, and involves an overall analysis of the scientific process to identify the areas that can be improved. In this article by Bruce Booth, he reviews “Begley’s six rules”. Two of these rules focus on the controls and reagents used in the experiment.

Of equal importance are the instruments used to make measurements. For example, how often are the pipettes calibrated? Are all lab members adequately trained in technique? This chart from Gilson is a useful one to have i ...

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