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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.

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.

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.

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.

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.

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.

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 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

Brightness Is In The Eye Of The Detector - What To Consider When Designing Your Panel

Brightness Is In The Eye Of The Detector - What To Consider When Designing Your Panel

By: Tim Bushnell, PhD

The heart and soul of the flow cytometry experiment is the ‘panel.’ The unique combinations of antibodies, antigens, fluorochromes, and other reagents are central to identifying the cells of interest and extracting the data necessary to answer the question at hand. Designing the right panel for flow cytometry is essential for detecting different modalities. The more parameters that can be interrogated will yield more information about the target cells. Current instruments can measure as many as 40 different parameters simultaneously. This is exciting, as it allows for more complex questions to be studied. Panel design is also valuable for precious samples,…

7 Key Image Analysis Terms For New Microscopist

7 Key Image Analysis Terms For New Microscopist

By: Heather Brown-Harding, PhD

As scientists, we need to perform image analysis after we’ve acquired images in the microscope, otherwise, we have just a pretty picture and not data. The vocabulary for image processing and analysis can be a little intimidating to those new to the field. Therefore, in this blog, I’m going to break down 7 terms that are key when post-processing of images. 1. RGB Image Images acquired during microscopy can be grouped into two main categories. Either monochrome (that can be multichannel) or “RGB.” RGB stands for red, green, blue – the primary colors of light. The cameras in our phones…

5 Essential Concepts In Genome Assembly From NGS data

5 Essential Concepts In Genome Assembly From NGS data

By: Deepak Kumar, PhD

The main goal for researchers, clinicians, and students who perform Next Generation Sequencing (NGS) and produce sequenced data for diverse projects involving human samples is to find biomarkers or variants to make diagnoses; and deduce the genetic anomalies that could be responsible for the disease they are conducting research on. Most projects (academic or non-academic) constitute the prior ideology on deciphering the “unknown.” There are well-versed computational protocols and pipelines formulated by labs across the world in determining what the “unknown” variants are. The fact that we have the “reference” human genome available – thanks to the Human Genome Project – plays…

Tools to Improve Your Panel Design – Determining Antigen Density

Tools to Improve Your Panel Design – Determining Antigen Density

By: Tim Bushnell, PhD

When a researcher chooses to use flow cytometry to answer a scientific question, they first have to build a polychromatic panel that will take advantage of the power of the technology and experimental design. When we set up to use flow cytometry to answer a scientific question, we have to design a polychromatic panel that will allow us to identify the cells of interest – the target of the research.  To identify these cells, we need to build a panel that takes advantage of the relative brightness of the fluorochromes, the expression level of the different proteins on the cell,…

The 5 Essentials To Successful Spectral Unmixing

The 5 Essentials To Successful Spectral Unmixing

By: Heather Brown-Harding, PhD

In an ideal world, we would be able to use fluorophores that don’t have any overlap in emission spectra and autofluorescence wouldn’t obscure your signal. Unfortunately, we don’t live in such a world and often have to use two closely related dyes – or contend with fluorescent molecules that are innately part of our sample. Fluorescent molecules include chlorophyll, collagen, NADPH, and vitamin A.  One example that I recently encountered was developing a new probe for lipids. The reviewers requested a direct comparison of the new dye to Nile Red in the same sample. Both dyes would localize to the…

The Essential Dos and Don'ts of NGS (Next Generation Sequencing)

The Essential Dos and Don'ts of NGS (Next Generation Sequencing)

By: Deepak Kumar, PhD

Next Generation Sequencing (NGS) is a rapidly evolving and widely used method worldwide in both academic and non-academic settings. One of the most valuable aspects of NGS is producing millions of sequenced reads with diverse read lengths from small amounts of input DNA. NGS methods are extremely versatile; producing reads as short as 75 bp, as seen in SOLiD sequencing, to long reads ranging upwards of 1000bp in the case of Pyrosequencing.  Both long and short reads fill a unique niche for researchers. Longer reads generated from NGS are excellent for genomic rearrangement and genome assembly projects; especially when there…

The 5 Fundamental Methods For Imaging Nucleic Acids

The 5 Fundamental Methods For Imaging Nucleic Acids

By: Heather Brown-Harding, PhD

There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This blog will focus on recommendations for electrostatic sorters.

This Is How Full Spectrum Cytometry Works

This Is How Full Spectrum Cytometry Works

By: Tim Bushnell, PhD

There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This blog will focus on recommendations for electrostatic sorters.

My Proven 5-Point Fast Track To A Career In Flow

My Proven 5-Point Fast Track To A Career In Flow

By: Tim Bushnell, PhD

There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This blog will focus on recommendations for electrostatic sorters.

Up Your Stain Game With These 7 Non-Fluorescent Histology Dyes

Up Your Stain Game With These 7 Non-Fluorescent Histology Dyes

By: Heather Brown-Harding, PhD

There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This blog will focus on recommendations for electrostatic sorters.

3 Ways Flow Cytometry Can Be Used To Research Bacteria

3 Ways Flow Cytometry Can Be Used To Research Bacteria

By: Tim Bushnell, PhD

There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This blog will focus on recommendations for electrostatic sorters.

Avoid Flow Cytometry Faux Pas: How To Set Voltage The Right Way

Avoid Flow Cytometry Faux Pas: How To Set Voltage The Right Way

By: Tim Bushnell, PhD

There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This blog will focus on recommendations for electrostatic sorters.

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Top Technical Training eBooks

Get the Advanced Microscopy eBook

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.