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Flow Cytometry

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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2015 Staff Picks: Flow Cytometry

There’s a joke in here about our editorial team going with the flow, but I can’t quite seem to make it work (feel free to leave suggestions in the comments). Despite my inability to make a joke count, I do hope that you find these articles amusing and useful. Jen Redig, Managing Editor Spot the…

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Locating Your Cellular Apoptosis Squad: Annexin V Staining Assays

In real life, cells are instructed to commit suicide for the greater good of the organism. The programmed cell death (apoptosis) is important during development of a multi-cellular organism. A good example you will appreciate is the dis-appreance of the tail from a tadpole as it turns into a frog. On the reverse, the lack…

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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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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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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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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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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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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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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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