5 – Flow Cytometry

2020 - A Year Turned Upside Down

By: Tim Bushnell, PhD

What an incredible year 2020 has been. It started off like any other year and bam SARS-CoV-2 (aka COVID 19) entered the equation, bringing chaos and havoc to the world. Things kept changing overnight as new rules and regulations popped up. Masking, quarantine, and flatten the curve became common words in the news. How we met, how we interacted changed almost overnight. Throughout all of this, as we look to 2021, there is hope and optimism. Multiple vaccines have been developed, building on years of research into the SARS-CoV virus, with some approved for human use, and others on the horizon.…

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,…

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,…

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…

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…

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…

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…

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…

Discover The Myriad Applications Of Beads In Flow Cytometry

By: Tim Bushnell, PhD

What is the single-most important feature of a flow cytometry experiment? Arguably, it’s the stained cells that gather data about biological processes of interest. However, a flow cytometer can measure cell-like particles as well as cells, which opens the realm of cytometry to the use of microspheres. Most researchers are familiar with the 4-Cs that beads can be used for: Control, Calibration, Compensation, and Counting. Beyond the 4-Cs, many are familiar with the multiplex bead assays for measuring analytes. Today, we will take a look beyond these well-known uses and discover the myriad applications of the “Mighty Microspheres.”

Mass Cytometry Revolves Around These 5 Things

By: Tim Bushnell, PhD

Because mass cytometry allows users to characterize masses so effectively, data can be normalized much more efficiently than what traditional fluorescent flow will permit. If there is no working CyTof at your institution, you can still partner with CyTof-friendly research institutions that have the technology on hand. And because the samples are fixed, you can ship them overnight. This way, they will be analyzed for you. Today’s article will summarize the functionality of mass cytometry technology. This tech has been commercialized largely by Fluidigm in the CyTof systems. There are 5 key points to cover, or takeaways, that cytometrists should…

3 Ways To Improve Flow Cytometry Troubleshooting

By: Tim Bushnell, PhD

A lot of the troubleshooting is focused on fluidics issues. If you sit down and think about your workflow, and how you might want to add a couple of little tweaks here and there which will ultimately help you improve the quality of your data as well as aid you in identifying issues before they become problems your troubleshooting will be much smoother. Consider these three things, what do you before you start collecting data, ensure you have appropriate plots of time vs fluorescence for each of the lasers your using and apply appropriate gating procedures.

3 Requirements For Accurate Flow Cytometry Compensation

By: Tim Bushnell, PhD

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…