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Discover The Myriad Applications Of Beads In Flow Cytometry

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.”

5-Point Guide To Buying A New Microscope For Your Lab

5-Point Guide To Buying A New Microscope For Your Lab

By: Heather Brown-Harding, PhD

Have you ever noticed how painful it can be to purchase a new microscope? It would be hard to miss – this can be a frustrating process. A lot of scientists and students consider the new microscope hunt quite scary for a variety of reasons. It might be that you’re worried you won’t get the right microscope and that you’ll regret it, or you may find that dealing with salespeople, in general, makes you kind of uncomfortable. But remember, salespeople are just human beings like you and me, and if we can treat them as such, the whole process of…

5 Techniques For Dramatic Improvements In Reproducibility

5 Techniques For Dramatic Improvements In Reproducibility

By: Heather Brown-Harding, PhD

It’s not easy to improve reproducibility in your experiments. Image manipulation has become a major problem in science, whether intentional or accidental. This has exploded with the advent of digital imaging and software like Photoshop. There are even mobile applications like Instagram filters that can be used for imaging trickery. It should go without saying that image reuse/manipulation represents profound dishonesty in science – a field intended to uphold the most stringent possible standards of truthful inquiry! But what about studies with a sloppy or stunted capacity for reproduction? These, too, plague science and hinder our ability to seamlessly move…

3 Compensation Mistakes That Will Ruin Your Flow Cytometry Experiments

3 Compensation Mistakes That Will Ruin Your Flow Cytometry Experiments

By: Tim Bushnell, PhD

Compensation is necessary due to the physics of fluorescence. Basically, compensation is the mathematical process of correcting spectral spillover from a fluorochrome into a secondary detector so that it is possible to identify single positive events in the context of a multidimensional panel. Good compensation requires that your controls tightly adhere to three rules. If the controls don’t meet this criteria, it will lead to faulty compensation resulting in false conclusions and poorly reproducible data. Even among flow cytometry veterans, a strong foundation is occasionally in need of a tune-up. And in a topic as complex as flow cytometry, it’s important that we review the fundamentals on a regular basis. In fact, it is the longtime cytometry expert who must check themselves for any sort of faith in older compensation practices. Science is ever a work in progress, and traditional methods are not always the right methods.

7 Individual Artifacts In Fluorescence Microscopy And How To Minimize Them

7 Individual Artifacts In Fluorescence Microscopy And How To Minimize Them

By: Heather Brown-Harding, PhD

There are 7 different common “artifacts” that may be affecting the quality of your imaging. Before digging into the details, let’s begin by defining an artifact: Essentially, it is any error introduced through sample preparation, the equipment or post-processing methods. This is an important concept to grasp because the effects can cause false positives or negatives, and they can physically distort your data. This is, of course, at odds with your mission to obtain reliable quantitative data. So what can you do to stop these artifacts? The problems can range from dirty objectives to bigger issues like light path aberrations.

We Tested 5 Major Flow Cytometry SPADE Programs for Speed - Here Are The Results

We Tested 5 Major Flow Cytometry SPADE Programs for Speed - Here Are The Results

By: Tim Bushnell, PhD

In the flow cytometry community, SPADE (Spanning-tree Progression Analysis of Density-normalized Events) is a favored algorithm for dealing with highly multidimensional or otherwise complex datasets. Like tSNE, SPADE extracts information across events in your data unsupervised and presents the result in a unique visual format. Given the growing popularity of this kind of algorithm for dealing with complex datasets, we decided to test the SPADE algorithm in 5 software packages, including Cytobank, FCS Express, FlowJo, R, and the original, free software made available by the author of SPADE. Which was the fastest?

Mass Cytometry Revolves Around These 5 Things

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 keep in mind as they perform their research. The 5 points include how mass cytometry works, panel design, proper sample preparation, data analysis, and imaging mass cytometry.

Use These 5 Techniques for Super Resolution

Use These 5 Techniques for Super Resolution

By: Heather Brown-Harding, PhD

When you need better resolution than what can be achieved using a traditional microscope, it can be very intimidating to figure out which machines will work best for your experiment. Super-resolution imaging methods require software reconstruction after image acquisition. This is because multiple images are required, and they need to be combined. Additionally, the points of light need to be reassigned to their true location. Today, we're going to discuss 5 different super resolution methods their pros and cons. Although Rayleigh Criterion is not broken, these techniques each feature creative ways to get around it.

Understanding Reproducibility in Flow Cytometry - It’s the Antibodies!

Understanding Reproducibility in Flow Cytometry - It’s the Antibodies!

By: Tim Bushnell, PhD

Reproducibility is key to the scientific method. After the results of a study are published, the community validates the findings and extends them. If the findings are not reproducible, the second step is impossible. With performable experiments increasing in complexity, and the concurrent increase in the cost of equipment and reagents to perform these experiments, it is important to find the best way to maximize the money spent on advancing research. In flow cytometry, there are many places where improvements can be made to increase the consistency and reproducibility of an experiment. The most obvious place is in the instrument, but today’s focus is on the reagents we use to identify cells of interest: Antibodies and fluorochromes.

3 Components Of Every Flow Cytometer You Don’t Know Enough About

3 Components Of Every Flow Cytometer You Don’t Know Enough About

By: Tim Bushnell, PhD

All flow cytometer instruments have a certain 3 components, and the way they are put together will dictate the performance of the system. As a user, you’ll be interacting heavily with these components, so you need to know both what they are and how they work. There are fluidics, optics, and electronics. The fluidics allow you to interact at the right flow rate so that your data keep a tight CV. Then you can run the same flow rate for all your samples, and you won't have different CVs for different samples. There are also different optics you can use, like PMTs, APDs, and PDs. It's important to remember the bandpass filters because they indicate the detector on which your signal will be measured. And with a newer generation of instruments, you can actually change out bandpass filters and design the flow cytometer to your specifications - just make sure you cite the specific bandpass filter that you use. Finally, there are electronics, which process the photon into an electronic signal that is ultimately digitized and stored in a file known as the “FCS file.” An analysis can be performed on this file at a later time.

4 Ways To Analyze Tissues By Flow Cytometry

4 Ways To Analyze Tissues By Flow Cytometry

By: Tim Bushnell, PhD

Did you know that tissues can be measured by flow cytometry? Flow cytometry is the measurement of cellular processes at the whole-cell level. This definition is useful because it includes not only flow cytometry, but any technique that measures at the level of the whole cell. Microscopy, for instance, is a great example of cytometry. But, what can be measured by flow cytometry? For one, tissues with lots of cells. When flow cytometry is practiced, the cells are broken up. Therefore, any cellular interactions within the sample are also broken up. This includes tissues, cell-to-cell contacts in tissues, and virtually any information about the microenvironment. As we continue to discover, the microenvironment can play a dramatic role in cell development, influencing how cells grow and change. This article will discuss how to analyze tissues and microenvironments by flow cytometry.

Ask These 7 Questions Before Purchasing A Flow Cytometer

Ask These 7 Questions Before Purchasing A Flow Cytometer

By: Tim Bushnell, PhD

I am still convinced that my first cell sorter was possessed. The number of issues that I had with the system remains hard for me to believe, even after all these years. It had been purchased, in part, from one vendor because the sales rep for a competitor was nowhere to be found. At that time, I admit I wasn’t overly diligent in my research process. Since then, I’ve pinpointed some critical questions that need to be answered before purchasing a new instrument. At the end of the process, a shiny new instrument will arrive at your facility. Make sure you find time to do a shakedown and validate the system. This is the time to get to know it better, identify quirks and potential issues, and develop training and QC programs. Once your shakedown is complete, you can start adding users and encouraging feedback on the system.

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.