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How to Perform Doublet Discrimination In Flow Cytometry

How to Perform Doublet Discrimination In Flow Cytometry

By: Tim Bushnell, PhD

You are probably familiar with the term, “doublet discrimination” or “doublet exclusion”, and have likely included this flow cytometry measurement into at least some (if not all) of your gating strategies. Even though you may utilize this important gating strategy, you may not have had the chance to delve deeper to explore exactly what doublets are and why it’s critical to exclude them. This article aims to give you insight on the what, why, and how of doublet discrimination.

3 Flow Cytometry Gates That Will Improve The Accuracy Of Your FACS Data Analysis

3 Flow Cytometry Gates That Will Improve The Accuracy Of Your FACS Data Analysis

By: Tim Bushnell, PhD

When training new users on data analysis, there are several different best practices and gating strategies you should incorporate into your analysis. There are also several misconceptions you must understand. There are 3 gates that many researchers are not using but should be using when analyzing their flow cytometry data. These gates are critical for good data analysis. They will help remove many confounding events that may be clouding your analysis, especially where rare events are concerned.

When To Use (And Not Use) Flow Cytometry Isotype Controls

When To Use (And Not Use) Flow Cytometry Isotype Controls

By: Tim Bushnell, PhD

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 everything you need to know about using (or not using) isotype controls in your next flow cytometry experiment.

How To Analyze FACS Data And Prepare Flow Cytometry Figures For Scientific Papers

How To Analyze FACS Data And Prepare Flow Cytometry Figures For Scientific Papers

By: Tim Bushnell, PhD

When preparing figures for publication, the scientific question and hypothesis that forms the basis of the paper must be central and all the figures must be in support of that. The flow cytometry data that forms the basis of the conclusions should be presented clearly and concisely. While it provides pretty pictures and colorful layouts, the meat of the data are the numbers ― percentages of populations, fluorescent intensity levels and the like ― are what will convince the reader that the hypothesis tested is valid and well thought out. Here’s how to choose the correct flow figure for presenting your data.

5 Gating Strategies To Get Your Flow Cytometry Data Published In Peer-Reviewed Scientific Journals

5 Gating Strategies To Get Your Flow Cytometry Data Published In Peer-Reviewed Scientific Journals

By: Tim Bushnell, PhD

When sitting down to perform a new analysis of flow cytometry data, the researcher is guided by very particular laws of nature and a very specific method of working through a biological hypothesis to avoid shaping the results to his or her whims. Following these 5 data analysis and gating strategies through the hierarchy described in this article, researchers are provided with several strategies for identifying and displaying the most relevant data from their flow cytometry experiments.

5 Essential Calculations For Accurate Flow Cytometry Results

5 Essential Calculations For Accurate Flow Cytometry Results

By: Tim Bushnell, PhD

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.

How To Set And Monitor Optimal Voltages For A Flow Cytometry Experiment

How To Set And Monitor Optimal Voltages For A Flow Cytometry Experiment

By: Tim Bushnell, PhD

The best way to take out the fear and agony of setting voltages is to use some optimization methods. The peak 2 method is a useful and robust method of identifying optimal PMT voltage ranges. Refining that to the voltage walk with the actual cells and fluorochromes of interest will further improve sensitivity, which is especially critical for rare cell populations or emergent antigens. This article describes how to set up, monitor, and maintain optimal voltage settings for your flow cytometry experiment.

What Is A Fluorescence Minus One, or FMO Control

What Is A Fluorescence Minus One, or FMO Control

By: Tim Bushnell, PhD

The Fluorescence Minus One Control, or FMO control is a type of control used to properly interpret flow cytometry data.  It is used to identify and gate cells in the context of data spread due to the multiple fluorochromes in a given panel. An FMO control contains all the flurochromes in a panel, except for the one that is being measured.  For example, in the four color panel, there would be four separate FMO controls, as shown in the table below. The FMO control ensures that the any spread of the fluorochromes into the channel of interest is properly identified.…

4 Biggest Mistakes Scientists Make During Multicolor Flow Cytometry Cell Sorting Experiments

4 Biggest Mistakes Scientists Make During Multicolor Flow Cytometry Cell Sorting Experiments

By: Tim Bushnell, PhD

Multicolor sorting experiments can be complicated and if not setup properly, result in wasted time and suboptimal results. When setting up a multicolor experiment, the most saliently critical step is to set PMT voltages properly. In addition, using a viability dye and addressing doublet discrimination and setting the right sort regions and gates is important for any kind of flow cytometry experiment, but particularly for cell sorting. These tips help to ensure your setup is perfect to achieve results of the highest caliber.

Why You Need To Use FMO Controls For All Multicolor Flow Cytometry Experiments

Why You Need To Use FMO Controls For All Multicolor Flow Cytometry Experiments

By: Tim Bushnell, PhD

FMO controls are samples that contain all the antibodies you are testing in your experimental samples, minus one of them. When analyzing the minus, or left out parameter in an FMO control, you give yourself a strong negative control to work with. It’s a strong negative control because the left out marker in the FMO control allows you to take into account how the other stains in your panel affect the respective minus parameter. Many flow cytometry gates are difficult to define. This is especially true when you’re looking at activation markers within a continuum or accounting for the large data spread that occurs when compensating a 10+ color experiment. The only way to convince reviewers that your gate is in the proper place is by using FMO controls. Here's why you need to use FMO controls for any multicolor flow cytometry experiment and how to prepare these controls properly.

What Is Flow Cytometry Light Scatter And How Cell Size And Particle Size Affects It

What Is Flow Cytometry Light Scatter And How Cell Size And Particle Size Affects It

By: Tim Bushnell, PhD

Forward scatter detectors collect light at small angles relative to the incident beam and can take advantage of the fact that cells preferentially scatter light in this “forward” direction. Forward scattered light is traditionally and often effectively measured with a photodiode, rather than the more sensitive photomultiplier used to measure fluorescence and side scatter. Scatter gets dim very quickly when particles have diameters below the wavelength of illuminating light, considering that scatter intensity decreases with a dependence on r6 of the particle. Here’s how small particles affect light scatter.

What Is Sheath Fluid

What Is Sheath Fluid

By: Tim Bushnell, PhD

Sheath fluid is the solution that runs in a flow cytometer.  Once the sheath fluid is running at laminar flow, the cells are injected into the center of the stream, at a slightly higher pressure.  The principles of hydrodynamic focusing cause the cells to align, single file in the direction of flow. Depending on experimental needs, different formulations of sheath fluid can be used. Many labs purchase pre-mixed phosphate-buffered saline from Leinco Technologies. Some researchers use Hepes-buffered saline.  This is particularly useful for high-pressure cell sorting as Hepes controls pH better at high pressure than phosphate buffers do. Finally, since…

Recent Articles

The Power Of Spectral Viewers And Their Use In Full Spectrum Flow Cytometry

The Power Of Spectral Viewers And Their Use In Full Spectrum Flow Cytometry

By: Tim Bushnell, PhD

What photon from yonder fluorochrome breaks?  It is … umm… hmmm. Let me see. Excitation off a 561 nm laser, with an emission maximum of 692 nm. I’m sure if Shakespeare was a flow cytometrist, he might have written that very scene. But the play is lost in time. However, since the protagonist had difficulty determining what fluorochrome was emitting photons, let’s consider how this could be figured out. In my opinion, one of the handiest flow cytometry tools is the spectral viewer. This tool helps visualize the excitation and emission profile of different fluorochromes, as well as allowing you…

Fickle Markers: Solutions For Antibody Binding Specificity Challenges

Fickle Markers: Solutions For Antibody Binding Specificity Challenges

By: Tim Bushnell, PhD

Reproducibility has been an ongoing, and important, concept in the sciences for years.  In the area of biomedical research, the alarm was sounded by several papers published in the early 2010’s.  Authors like Begley and Ellis, Prinz and coworkers, and Vasilevsky and colleagues, among others reported an alarming trend in the reproducibility of pre-clinical data.  These reports indicated between 50% to almost 90% of published pre-clinical data were not reproducible.  This was further highlighted in the article by Freedman and coworkers, who tried to identify and quantify the different sources of error that could be causing this crisis.  Figure 1,…

5 Common Flow Cytometry Questions, Answered

5 Common Flow Cytometry Questions, Answered

By: Tim Bushnell, PhD

I want to thank all of you who send us your questions about flow cytometry, so I thought I would dip into the old email bag and answer a few of the common ones here.  If your question isn’t answered this time, look for it to be answered in a future blog post.  Of course, if you want us to cover a specific topic, drop us a line.  1. How Fast Can I Go? This is  a common question. The allure of the ‘hi’ button is hard to resist.  The faster you go, the sooner you are finished with data…

Combining Flow Cytometry With Plant Science, Microorganisms, And The Environment

Combining Flow Cytometry With Plant Science, Microorganisms, And The Environment

By: Tim Bushnell, PhD

My first introduction to flow cytometry was talking to a professor who’d brought one on a research cruise to study phytoplankton. It was only later that I was introduced to the marvelous world that’s been my career for over 20 years.   In that time, I’ve had the opportunity to work with researchers in many different areas, exposing me to a wide variety of cell types and more important assays. What continues to amaze me is the number of different parameters we can measure, not just the number of fluorochromes, but the information we can extract from samples – animal, vegetable…

Common Numbers-Based Questions I Get As A Flow Cytometry Core Manager And How To Answer Them

Common Numbers-Based Questions I Get As A Flow Cytometry Core Manager And How To Answer Them

By: Tim Bushnell, PhD

Numbers are all around us.  My personal favorite is ≅1.618 aka ɸ aka ‘the golden ratio’.  It’s found throughout history, where it has influenced architects and artists. We see it in nature, in plants, and it is used in movies to frame shots. It can be approximated by the Fibonacci sequence (another math favorite of mine). However, I have not worked out how to apply this to flow cytometry.  That doesn’t mean numbers aren’t important in flow cytometry. They are central to everything we do, and in this blog, I’m going to flit around numbers-based questions that I have received…

3 Must-Have High-Dimensional Flow Cytometry Controls

3 Must-Have High-Dimensional Flow Cytometry Controls

By: Tim Bushnell, PhD

Developments such as the recent upgrade to the Cytobank analysis platform and the creation of new packages such as Immunocluster are reducing the computational expertise needed to work with high-dimensional flow cytometry datasets. Whether you are a researcher in academia, industry, or government, you may want to take advantage of the reduced barrier to entry to apply high-dimensional flow cytometry in your work. However, you’ll need the right experimental design to access the new transformative insights available through these approaches and avoid wasting the considerable time and money required for performing them. As with all experiments, a good design begins…

The Fluorochrome Less Excited: How To Build A Flow Cytometry Antibody Panel

The Fluorochrome Less Excited: How To Build A Flow Cytometry Antibody Panel

By: Tim Bushnell, PhD

Fluorochrome, antibodies and detectors are important. The journey of a thousand cells starts with a good fluorescent panel. The polychromatic panel is the combination of antibodies and fluorochromes. These will be used during the experiment to answer the biological question of interest. When you only need a few targets, the creation of the panel is relatively straightforward. It’s only when you start to get into more complex panels with multiple fluorochromes that overlap in excitation and emission gets more interesting.  FLUOROCHROMES Both full spectrum and traditional fluorescent flow cytometry rely on measuring the emission of the fluorochromes that are attached…

Flow Cytometry Year in Review: Key Changes To Know

Flow Cytometry Year in Review: Key Changes To Know

By: Meerambika Mishra

Here we are, at the end of an eventful year 2021. But with the promise of a new year 2022 to come. It has been a long year, filled with ups and downs. It is always good to reflect on the past year as we move to the future.  In Memoriam Sir Isaac Newton wrote “If I have seen further, it is by standing upon the shoulders of giants.” In the past year, we have lost some giants of our field including Zbigniew Darzynkiwicz, who contributed much in the areas of cell cycle analysis and apoptosis. Howard Shapiro, known for…

What Star Trek Taught Me About Flow Cytometry

What Star Trek Taught Me About Flow Cytometry

By: Tim Bushnell, PhD

It is no secret that I am a very big fan of the Star Trek franchise. There are many good episodes and lessons explored in the 813+ episodes, 12 movies (and counting). Don’t worry, this blog is not going to review all 813, or even 5 of them. Instead, some of the lessons I have taken away from the show that have applicability to science and flow cytometry.  “Darmok and Jalad at Tanagra.”  (ST:TNG season 5, episode 2) This is probably one of my favorite episodes, which involves Picard and an alien trying to establish a common ground and learn…

5 Flow Cytometry Strategies That Sun Tzu Taught Me

5 Flow Cytometry Strategies That Sun Tzu Taught Me

By: Tim Bushnell, PhD

Sun Tzu was a Chinese general and philosopher. His most famous writing is ‘The Art of War’, and has been studied by generals and CEOs, to glean ideas and strategies to help their missions. I was recently rereading this work and thought to myself if any of Sun Tzu’s lessons could apply to flow cytometry.  So I have identified 5 points that I think lend themselves to thinking about flow cytometry.  “Quickness is the essence of the war.” In flow cytometry, speed is of the essence. The longer the cells are out of their natural environment, the less happy they…

A Basic Guide To Flow Cytometry (3 Foundational Concepts)

A Basic Guide To Flow Cytometry (3 Foundational Concepts)

By: Meerambika Mishra

Mastering foundational concepts are imperative for successfully using any technique or system.  Robert Heinlein introduced the term ‘Grok’  in his novel Stranger in a Strange Land. Ever since then it has made its way into popular culture. To Grok something is to understand it intuitively, fully. As a cytometrist, there are several key concepts that you must grok to be successful in your career. These foundational concepts are the key tools that we use day in and day out to identify and characterize our cells of interest.  Cells Flow cytometry measures biological processes at the whole cell level. To do…

4 Critical Rules For Spectral Unmixing

4 Critical Rules For Spectral Unmixing

By: Tim Bushnell, PhD

Spectral unmixing is the mathematical process by which a spectrum is broken down into the abundances of the different fluorochromes that make up the observed spectrum. This was described in the paper by Novo et al., (2013), which presented a generalized model for spectral unmixing of flow cytometry data. Of course, like compensation in traditional fluorescent flow cytometry, there are important rules to observe regarding the controls that are used to unmix the sample. If you need a refresher on the rules for TFF compensation, you can read about them here.    This blog will discuss the generalized process of spectral unmixing…

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