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Featured Articles

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.

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.…

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.

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.

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.

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.

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.

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.

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.

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.

How To Differentiate T-Regulatory Cells (Tregs) By Flow Cytometry

How To Differentiate T-Regulatory Cells (Tregs) By Flow Cytometry

By: Tim Bushnell, PhD

T regulatory cells (Tregs), formerly known as T suppressor cells, are a T cell subset with direct roles in both autoimmunity and responses to pathogens. Tregs decrease inflammation via the secretion of immunosuppressive cytokines (IL-10, TGF-b) and also through direct suppression of inflammatory effector T cells (such as Th1 and Th17 cells). Given the importance of this unique T cell subset in so many immune responses, many investigators feel remiss if they immunophenotype their cell populations of interest without including a Treg measurement in the mix. But quantifying Tregs can be complicated. This article will show you how to quantify Tregs and how to ensure you're measuring true suppressor T cells.

Recent Articles

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…

How To Buy A Flow Cytometer - What You Need To Evaluate From A To Z

How To Buy A Flow Cytometer - What You Need To Evaluate From A To Z

By: Tim Bushnell, PhD

So you have the money to buy a flow cytometer. Is it a sorter? Or perhaps a spectral analyzer? No wait, maybe an imaging mass cytometer?  Big or small?  What to choose?  How to choose?  More importantly, once you sign the contract to purchase the instrument, you don’t want to be struck with buyers remorse.  It is indeed a big decision and we have the best advice for you to consider before making the purchase. Let’s discuss some of the steps you should take to prevent buyers remorse and ensure you are getting the best instrument for your needs.  Do…

How To Do Variant Calling From RNASeq NGS Data

How To Do Variant Calling From RNASeq NGS Data

By: Deepak Kumar, PhD

Developing variant calling and analysis pipelines for NGS sequenced data have become a norm in clinical labs. These pipelines include a strategic integration of several tools and techniques to identify molecular and structural variants. That eventually helps in the apt variant annotation and interpretation. This blog will delve into the concepts and intricacies of developing a “variant calling” pipeline using GATK. “Variant calling” can also be performed using tools other than GATK, such as FREEBAYES and SAMTOOLS.  In this blog, I will walk you through variant calling methods on Illumina germline RNASeq data. In the steps, wherever required, I will…

How small can you go? Flow cytometry of bacteria and viruses

How small can you go? Flow cytometry of bacteria and viruses

By: Tim Bushnell, PhD

Flow cytometers are traditionally designed for measuring particles, like beads and cells. These tend to fall in the small micron size range. Looking at the relative size of different targets of biological interest, it is clear the most common targets for flow cytometry (cells) are comparatively large (figure 1). Figure 1:  Relative size of different biological targets of interest. Image modified from Bioninja.    In the visible spectrum, where most of the excitation light sources reside, it is clear the cells are larger than the light. This is important as one of the characteristics that we typically measure is the amount…

What Is Spectral Unmixing And Why It's Important In Flow Cytometry

What Is Spectral Unmixing And Why It's Important In Flow Cytometry

By: Tim Bushnell, PhD

As the labeled cell passes through the interrogation point, it is illuminated by the excitation lasers. The fluorochromes, fluoresce; emitting photons of a higher wavelength than the excitation source. This is typically modeled using spectral viewers such as in the figure below, which shows the excitation (dashed lines) and emission (filled curves) for Brilliant Violet 421TM (purple) and Alexa Fluor 488Ⓡ (green).  Figure 1: Excitation and emission profiles of BV421TM and AF488Ⓡ  In traditional fluorescent flow cytometry (TFF), the instrument measures each fluorochrome off an individual detector. Since the detectors we use — photomultiplier tubes (PMT) and avalanche photodiodes (APD)…

How To Extract Cells From Tissues Using Laser Capture Microscopy

How To Extract Cells From Tissues Using Laser Capture Microscopy

By: Tim Bushnell, PhD

Extracting specific cells still remains an important aspect of several emerging genomic techniques. Prior knowledge about the input cells helps to put the downstream results in context. The most common isolation technique is cell sorting, but it requires a single cell suspension and eliminates any spatial information about the microenvironment. Spatial transcriptomics is an emerging technique that can address some of these issues, but that is a topic for another blog.  So what does a researcher who needs to isolate a specific type of cell do? The answer lies in the technique of laser capture microdissection (LCM). Developed at the National…

The Importance Of Quality Control And Quality Assurance In Flow Cytometry (Part 4 Of 6)

The Importance Of Quality Control And Quality Assurance In Flow Cytometry (Part 4 Of 6)

By: Tim Bushnell, PhD

Incorporating quality control as a part of the optimization process in  your flow cytometry protocol is important. Take a step back and consider how to build quality control tracking into the experimental protocol.  When researchers hear about quality control, they immediately shift their attention to those operating and maintaining the instrument, as if the whole weight of QC should fall on their shoulders.   It is true that core facilities work hard to provide high-quality instruments and monitor performance over time so that the researchers can enjoy uniformity in their experiments. That, however, is just one level of QC.  As the experimental…

Understanding Clinical Trials And Drug Development As A Research Scientist

Understanding Clinical Trials And Drug Development As A Research Scientist

By: Deepak Kumar, PhD

Clinical trials are studies designed to test the novel methods of diagnosing and treating health conditions – by observing the outcomes of human subjects under experimental conditions.  These are interventional studies that are performed under stringent clinical laboratory settings. Contrariwise, non-interventional studies are performed outside the clinical trial settings that provide researchers an opportunity to monitor the effect of drugs in real-life situations. Non-interventional trials are also termed observational studies as they include post-marketing surveillance studies (PMS) and post-authorization safety studies (PASS). Clinical trials are preferred for testing newly developed drugs since interventional studies are conducted in a highly monitored…

Which Fluorophores To Use For Your Microscopy Experiment

Which Fluorophores To Use For Your Microscopy Experiment

By: Heather Brown-Harding, PhD

Fluorophore selection is important. I have often been asked by my facility users which fluorophore is best suited for their experiments. The answer to this is mostly dependent on whether they are using a widefield microscope with set excitation/emission cubes or a laser based system that lets you select the laser and the emission window. Once you have narrowed down which fluorophores you can excite and collect the correct emission, you can further refine the specific fluorophore that is best for your experiment.  In this blog  we will discuss how to determine what can work with your microscope, and how…

How To Optimize Instrument Voltage For Flow Cytometry Experiments  (Part 3 Of 6)

How To Optimize Instrument Voltage For Flow Cytometry Experiments (Part 3 Of 6)

By: Tim Bushnell, PhD

As we continue to explore the steps involved in optimizing a flow cytometry experiment, we turn our attention to the detectors and optimizing sensitivity: instrument voltage optimization.  This is important as we want to ensure that we can make as sensitive a measurement as possible.  This requires us to know the optimal sensitivity of our instrument, and how our stained cells are resolved based on that voltage.  Let’s start by asking the question what makes a good voltage?  Joe Trotter, from the BD Biosciences Advanced Technology Group, once suggested the following:  Electronic noise effects resolution sensitivity   A good minimal PMT…

How To Profile DNA And RNA Expression Using Next Generation Sequencing (Part-2)

How To Profile DNA And RNA Expression Using Next Generation Sequencing (Part-2)

By: Deepak Kumar, PhD

In the first blog of this series, we explored the power of sequencing the genome at various levels. We also dealt with how the characterization of the RNA expression levels helps us to understand the changes at the genome level. These changes impact the downstream expression of the target genes. In this blog, we will explore how NGS sequencing can help us comprehend DNA modification that affect the expression pattern of the given genes (epigenetic profiling) as well as characterizing the DNA-protein interactions that allow for the identification of genes that may be regulated by a given protein.  DNA Methylation Profiling…

4 No Cost Ways To Improve Your Microscopy Image Quality

4 No Cost Ways To Improve Your Microscopy Image Quality

By: Heather Brown-Harding, PhD

Image quality is critical for accurate and reproducible data. Many people get stuck on the magnification of the objective or on using a confocal instead of a widefield microscope. There are several other factors that affect the image quality such as the numerical aperture of the objective, the signal-to-noise ratio of the system, or the brightness of the sample.  Numerical aperture is the ability of an objective to collect light from a sample, but it contributes to two key formulas that will affect your image quality. The first is the theoretical resolution of the objective. It is expressed with the…

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Top Technical Training eBooks

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Get the Advanced Microscopy eBook

Heather Brown-Harding, PhD

Learn the best practices and advanced techniques across the diverse fields of microscopy, including instrumentation, experimental setup, image analysis, figure preparation, and more.

Get The Free Modern Flow Cytometry eBook

Get The Free Modern Flow Cytometry eBook

Tim Bushnell, PhD

Learn the best practices of flow cytometry experimentation, data analysis, figure preparation, antibody panel design, instrumentation and more.

Get The Free 4-10 Compensation eBook

Get The Free 4-10 Compensation eBook

Tim Bushnell, PhD

Advanced 4-10 Color Compensation, Learn strategies for designing advanced antibody compensation panels and how to use your compensation matrix to analyze your experimental data.