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Easy-To-Forget Flow Fundamentals That Thwart Bad Science

Easy-To-Forget Flow Fundamentals That Thwart Bad Science

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.

Important Controls For Your Flow Cytometry Lab

Important Controls For Your Flow Cytometry Lab

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.

4 Factors To Improve Flow Cytometry Cell Sorting Speed

4 Factors To Improve Flow Cytometry Cell Sorting Speed

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.

Optical Tissue Clearing For Pristine Sample Preparation

Optical Tissue Clearing For Pristine Sample Preparation

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.

4 Flow Cytometry Assays For Monitoring Intracellular Processes

4 Flow Cytometry Assays For Monitoring Intracellular Processes

By: Tim Bushnell, PhD

The most common flow assay is undoubtedly immunophenotyping, in which fluorescently tagged antibodies are used to bind to cellular proteins. This allows you to determine the types of cells present. As long as there is a fluorescent reporter available, it is possible to measure biological processes using flow cytometry – especially in a phenotypically defined manner. Probably the most common of these assays is the calcium flux assay. And that is just the tip of the iceberg. In addition to calcium, it is possible to measure magnesium and zinc concentrations, reactive oxygen species, and even membrane potential using flow. Today, we’ll cover 4 assays that use a fluorescent reporter to measure their target, allowing researchers to challenge the cells and measure their response in real time.

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.

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.