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Flow Cytometry

The Nope-Nope-Nevers of Using a Flow Cytometer

There are some wonderful toys in the lab that enable us to open up a whole new world in science. One of those is a rather pricey and an incredibly sensitive laser-based apparatus capable of counting and sorting cells, detecting biomarkers, and engineering proteins: the flow cytometer. By propelling cells through the path of the…

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MIFlowCyt Guidelines: Helping You to Publish Your Flow Data

Wow, you’ve done it! Your experiment worked and your boss asked you to write it up for publication in your favorite journal. Where to start with presenting your flow data? Take a deep breath, help is in hand in the form of the MIFloCyt Guidelines. As with other scientific techniques, there are ‘Minimum Information about…

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Troubleshooting: No Events on Your Cytometer

It’s happened to us all, you are ready to run your samples on the cytometer and you can’t see your cells on the screen. Here are a few tricks to troubleshooting this: Cytometer vs. computer connection The different types of cytometers will need different orders for switching on the cytometer and computer. Some are cytometer…

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Sorting Large Cells and Materials by Flow Cytometry

Flow cytometers and cell sorters were designed with blood cells in mind. This means that commercial cell sorters are optimized for sorting cells typically smaller than about 20 µm in diameter. However, it turns out that many cell types, including those of mammals, are larger than 20 µm. So what are your options if you…

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An Introduction to Spectral Overlap and Compensation Protocols in Flow Cytometry

It strikes fear into the hearts of new cytometrists. Compensation. More fights have started over the proper way to compensate at meetings than anything else. This article will strive to shed some light on the principles of compensation, and equip you with the tools necessary to achieve compensation mastery for your research experiments. Compensation is…

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Ain’t no mountain high enough: Summit Software

The town of Fort Collins is situated in the foothills of the Rocky Mountains in Colorado and it is those mountains that give inspiration to the name of the Beckman Coulter acquisition program Summit. Summit software started out as the software to run the MoFlo (Modular Flow) high-speed cell sorter that was designed by Ger…

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Thresholding in Flow Cytometry – Why It Is Important

Flow Cytometry is a great way of seeing how many of your cells express a particular marker and how much of it is there. We do this by measuring fluorescence, but, as with all measuring systems, there will be signal that we are always trying to measure the above the noise. The signal that we…

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The Proper Way To Use The Sub-G1 Assay

The sub-G1 assay for measuring apoptosis is easy, rapid, reliable, reproducible, and cheap and is widely used. However, you need to understand the mechanics of the assay and the apoptotic process, otherwise you could over- or underestimate your apoptosis results!

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The Exciting (and Emitting) World of Fluorescence

Flow cytometry is a fluorescence-based technology, as is fluorescence microscopy and confocal microscopy. Fluorescence is fundamental to how a cytometer gathers data, but I am often surprised, as a core manager, at how little new users know about the process of fluorescence. So, this is where I always start the training process. Let’s get physical…

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Catching Greatness: Measuring Cellular Degranulation

One of the key characteristics of cytotoxic cells (i.e. CD8+ T cells, natural killer cells) is the presence of pre-formed cytoplasmic lysosomal granules. These structures house perforin and granzyme; two molecules that are essential for the lysis of target cells. Upon effector cell activation, granules are polarized toward the target cell and the contents are…

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An Introduction to Biomarkers

During the past decade or two, the field of biomarkers has witnessed immense development. The complexity of the field mirrors its multi-faceted applications, and it is easy to get confused with the different terms used in different contexts. Hopefully, this article will help make things clearer! What is a Biomarker? The word ‘biomarker,’ has more…

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5 Ways to Improve Your Fluorescent Protein Sorting

If, like many people, you use fluorescent proteins to view your transfection efficiency or your CRISPR gene editing, you can also isolate cells with different expression levels of fluorescent proteins. Here are 5 ways to improve your sorting experiment.

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Know the Players: Combinatorial, Single-Cell Approaches to Explore the Complexity of Biologic Systems

In this webinar, presented by Jonathan M. Irish, PhD, Assistant Professor, Department of Cancer Biology, Vanderbilt University and Manisha Ray, PhD, Single-cell analysis expert, Fluidigm Corporation, you will learn: Importance and relevance of single-cell approaches to cancer. Cytomic techniques for single-cell approaches . Quality controls for single-cell techniques. Abstract: We know that cellular heterogeneity in…

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Herzenberg and the Invention of the FACS Machine

The flow cytometer that we have all grown to know and love may have only come into its own in the 1990’s, but who would have known that the first cell sorter was invented as early as the 1950’s? With the recent death of one of the key developers of fluorescence activated cell sorting (FACS),…

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Being “Accuri-te” Through Cytometry: A Guide to Accuri C6 Software

A new lab toy to make it big in the last 5–10 years is the Accuri C6 cytometer (now under the BD umbrella), a low-cost instrument in comparison to the big boys. Lightweight, with a small footprint and straightforward maintenance, it’s often the cytometer of choice. It may be suitable for those labs that require…

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Putting Down a Marker in Flow Cytometry to Help Determine Positivity

In many biological experiments the question that a researcher wants to ask is – ‘do some or all of my cells express a particular protein?’ There are many ways of doing this, which you will be familiar with e.g. Western blotting, immunoprecipitation, microscopic examination of stained cells and even mass spectrometry. Using Flow Cytometry to…

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Setting Voltages for Optimal Sensitivity

After panel design, and titration of the reagents, the next most important step in flow cytometry is setting the proper voltages on the photomultipler tubes (PMTs). These detectors take the photons of light emitted by the fluorochromes on the cells and convert them to electrons, which ultimately become the voltage current that is digitized and…

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A Guide to Flow Cytometry Software: Becton Dickinson’s ‘Diva’

If you use a Becton Dickinson (BD) cytometer in your lab, the chances are you are acquiring your data using ‘Diva’ software. Diva software is used to acquire your cytometry data on LSRII, LSRFortessa, CantoII and Aria cell sorters. As well as acquiring your data using Diva software, you can also analyse your data after…

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The Basics of Intracellular Cytokine Staining

In this webinar you will learn: The theory of intracellular cytokine staining. Experimental design and sample preparation considerations. Common pitfalls of intracellular cytokine staining. Abstract: Cytokine analysis via intracellular staining is arguably one of the most important technical advances in the immunologist’s toolbox in the last 20 years. It allows us to study the output…

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FlowJo Software: how’s your FlowJo mojo?

Generally, once you’ve acquired your samples on a cytometer, the hard (but hopefully fun!) part of making sense of all the data begins. This can be increasingly complex depending on the number of cells, populations, parameters, and combinations. So where we use the dedicated software aligned to the analyser for acquisition, we frequently use alternative…

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Measuring Extracellular Vesicles by Flow Cytometry: Challenges and Prospects

In this webinar you will learn: Key features of extracellular vesicles (EVs). Fundamental principles regarding their measurement by flow cytometry. Calibrators and standards for EV measurement by flow cytometry. A new high-resolution flow cytometry approach to measuring EVs. Abstract: Extracellular vesicles (EVs) are small (~100 nm) membrane-bound vesicles released from the cell surface and intracellular…

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Basic Statistics for Flow Cytometrists – Part 1

Part of my job in running a core flow cytometry facility is to make sure that the experiments that my users run have been optimised. But that optimisation can be split up into several areas. The first area is experimental planning: What do you want to know? Can you do this by flow cytometry? And…

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10 Hints For Setting Up A Core Flow Cytometry Lab

If you are establishing a flow core facility or even setting up just one cytometer, then you need to know a few things before you plan your new lab and start looking at purchasing cytometers: Before You Start 1. Know Your Customer It’s really important to know if you are going to be setting up and…

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Data Spread and How to Measure It: the Coefficient of Variation (CV)

No matter how we make measurements, there will be variation (a spread of data). Take 100 people and ask them to guess your age and you will get a range of results: some will be too low (excellent!), some too high (not so good!). It is the same with any of our laboratory experiments –…

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Shapiro’s Laws of Flow Cytometry

Once you have been in the field of Flow Cytometry for a bit, the name Howard Shapiro will be familiar to you. However, for those who are new to Cytometry, you might not be aware of Dr Shapiro and his fabulous book ‘Practical Flow Cytometry’. Most Cytometrists have a paper copy of this bible of Flow…

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Importance of Antibody Titration in Flow cytometry

After designing a multicolor flow cytometry panel and securing the necessary cells and reagents, the process of optimization of the panel can begin. The first step in that optimization is titration of your antibodies. In this process, following a standard protocol to be used in the final analysis, you stain a known amount of cells with…

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Beginners Guide to Designing Your Own Polychromatic Flow Panel

“The greater the power, the more dangerous the abuse.” – Edmund Burke “With great power comes great responsibility.” – Uncle Ben (quoting Voltaire) While it’s unlikely that either of these speakers performed multi-dimensional flow cytometry, it is important to remember these quotes in the context of developing and implementing a good polychromatic flow panel.  More fluorochromes are…

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A Numbers Game: the ‘How’ and ‘Why’ of Counting Cells

“It is the weight, not numbers of experiments that is to be regarded.”  Isaac Newton Read any flow cytometry protocol and somewhere near the beginning will state something to the effect of ‘Place 1 million cells into a tube.’ The question is, faced with that special sample for THE experiment, how do you count cells…

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All sorted? Getting the best out of your cell sorting facility

If you are bringing cells to a core to be sorted there are a few things you can do to optimise your sorting and will help the core staff. Here are a few hints: 1) Know the size of your cells Do you know the size of your cells?  Not many people do and its one of…

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Immunophenotyping: Identifying Who’s Who in the Cellular World

Figuring out what’s what When studying cells and cell subsets (and cell sub-subsets, and so on!!) we need ways to identify and classify every single cell. This will allow us to individually analyse each population and, for example, help to discover their role in health and disease. A principal way we do this is by looking…

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Fixation and flow cytometry

Fixation is routinely used in histology and cytology Labs the world over as a way of keeping cells in stasis at a particular point to ensure that, by the time they are examined, they have not deteriorated. This is also something that we often want to do in flow cytometry experiments. It seems like a…

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Using Flow Cytometry for Fluorescence Resonance Energy Transfer

A marriage of sorts Fluorescence resonance energy transfer, or FRET, is often done using a microscope, which means it can be difficult to analyze large numbers of cells in one sitting. One way to overcome this, is by combining FRET with fluorescent-activated cell sorting (FACS), giving you a high-throughput method to screen for protein interactions…

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Where Are My Cells: Part 2

The golden rule of flow cytometry, especially cell sorting is: ‘Put good cells in and get good cells out’. When you sort you might not get good cells out and you may not get the numbers you were expecting. In my previous article I  touched on a few reasons why your cell numbers might be…

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Using Flow Cytometry for Cell Proliferation Assays: Tips for Success

We all know, generally from bitter experience, that experiments don’t always work first time and that sometimes the little things that govern success are the things that get left out of that online protocol! So whether you are assessing proliferation by nucleotide incorporation or by dye dilution, here are some handy hints to help you…

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Where Are My Cells: Part 1

If there are a million cells of interest in your sample and you pass them through a sorter, you might expect to get a million cells back. But you don’t – and here is why. Hardware aborts A hardware abort occurs when an instrument can’t process the information about an event because it is still…

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One For You, One For Me… How a Cell Sorter Works

Cell sorters do not operate by magic, even it looks that way. It’s about the application of physics, electronics, fast computers and formation of droplets. Whether you bring your cells to a flow core to be sorted or you sort them yourself, it important to know how the cell sorter works. Cells can be sorted…

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Crap in, crap out: Flushing Out The Problems in Your Flow Cytometry Data

“What Have You Done To My Cells??!!!” This cry of pain from researchers, frequently aimed at core facility operators, is heard after receiving incomprehensible data for an invaluable tube of cells. Equally baffling to the trained user of flow cytometric instrumentation is when data emerges that is either unreliable or inconsistent with the known properties…

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