Five Things That Irritate Flow Cytometrists

Five Things That Irritate Flow Cytometrists

I have worked in flow cytometry for a number of years. I’m still annoyed that many myths and imprecisions are perpetrated and perpetuated. Here is my non-exhaustive list of cytometry-related beliefs that send flow cytometrists screaming from the room or at least, being English, make me tut sadly. Forward Scatter Equals Cell Size No No…

Guidelines for Efficient Cell Sorting – Part 1

Flow cytometry is a pervasive tool to characterize just about anything in cell biology. From quantifying the expression of surface antigens, to determining the physiological changes in cells and everything in between, flow cytometry is as indispensable to a cell biologist as a knife is to a surgeon. Cell sorting is pivotal in enabling researchers…

Brefeldin A v Monensin: How to Hunt for Proteins

Brefeldin A v Monensin: How to Hunt for Proteins

As any good biologist knows, one of the easiest ways to determine if a cell is functionally active is the production and secretion of proteins in response to a stimulus. In many circumstances, the quantity of the secreted protein, and thus the level of cellular activation can be assessed by ELISA. However, if you are…

Chromosome Analysis by Flow Cytometry

Chromosome Analysis by Flow Cytometry

In most people’s minds a flow cytometer can sort, view and count cells e.g. lymphocytes, thymocytes, cultured cells and even non-mammalian cells such as yeast or bacteria. However, in reality, a flow cytometer is capable of providing information about any particle as long as it has detectable fluorescence. This fluorescence may occur either inherently or…

How to Store Your Reagents, so They ‘Do Exactly What It Says on the Tin’

How to Store Your Reagents, so They ‘Do Exactly What It Says on the Tin’

Your reagents should do ‘Exactly what they say on the tin.’  This only happens though if you look after them in the way the manufacturer states on their data sheets. We have all been guilty of using reagents past their expiration date.  Usually we can get away with it, but there are a few things…

Are Quantum Dots Any Good for Flow Cytometry?

Are Quantum Dots Any Good for Flow Cytometry?

What Are Quantum Dots? Quantum dots were discovered in the early 1980s. However, it was not until the late 1990s that their use in biological applications was suggested.1 Quantum dots are semiconducting nanocrystals made of artificial atom clusters. Their size generally ranges from 2 to 20 nm. Size is crucial for their physical properties because…

How to perform cell synchronization in specific cell cycle phases

How to perform cell synchronization in specific cell cycle phases

The cell cycle has been very well documented over the years because of its dysregulation in diseases such as cancer. Many different processes contribute to cell growth and replication, which is ultimately controlled by a series of tightly controlled cell cycle phases. For some areas of research, especially within drug discovery and cancer research, cell synchronization in…

Hierarchical or Boolean Gating: Which One to Choose?

Hierarchical or Boolean Gating: Which One to Choose?

A flow cytometer collects the events you are interested in, and also ‘sees’ every event that goes through. This includes debris and even bits in your buffers. As cytometrists, we gate our cells to exclude unwanted bits and to focus on the sub-populations that we are interested in studying. There are two main ways of gating…

How Fluorescent Molecules Work

How Fluorescent Molecules Work

Fluorescence is one of the most important and useful tools in a biologist’s toolbox. In biology, nearly every field, from physiology to immunology, uses fluorescent molecules (aka fluorophores) to detect proteins. However, the specific science behind how fluorescence works can be confusing or overlooked. Have no fear! In this article, we break down key points of…

Multiplex Cytometric Bead Array:  The ABCs of CBAs

Multiplex Cytometric Bead Array: The ABCs of CBAs

Multi-parameter data acquisition is key to the modern era of science research. I, for one, wish every single experiment that I design would give me the maximum amount of information. For example, in cell biology and immunology, we want to capture as much information (be it cytokines/hormones/chemokines) as possible about a given cell population. Of…

Demystifying the Flow Cytometry Optics System:  A Peek Under the Hood

Demystifying the Flow Cytometry Optics System: A Peek Under the Hood

To many users, the flow cytometer is a magic box: put in cells, get out data. You click the button to tell it which colors to look at without much thought about how the machine does this. However, not all fluorophores are created equal—some configurations might exclude the spectrum you’re really looking for. Here’s a…

Corralling Your Cells: How to Gate in Flow Cytometry

Corralling Your Cells: How to Gate in Flow Cytometry

Flow cytometry. Some people love it—most hate it—but all can agree that it is one of the most powerful analytical tools immunologists possess. Here’s a quick refresher: as the name suggests, flow cytometry measures the physical and chemical characteristics of cells. This is accomplished by fluorescently labeling cell surface markers/proteins using antibodies conjugated to fluorophores….

Lighting the Way: Understanding Flow Cytometry Fluorophores

Lighting the Way: Understanding Flow Cytometry Fluorophores

As science is becoming more interdisciplinary, the tools we use to answer questions are also crossing party lines. Case in point: flow cytometry. Once a tool only used by “real” immunologists, flow cytometry is fast becoming a method by which numerous questions can be answered, from the length of a cell’s telomeres, to the state…

Detection of Apoptosis by Flow Cytometry: To Be or Not to Be

Detection of Apoptosis by Flow Cytometry: To Be or Not to Be

Sometimes only a small subset of a cell population will show apoptotic features making flow cytometry an excellent way to identify and quantify them. A previous Bitesize Bio article showed how flow cytometry can detect apoptotic hallmarks. More than 30 different dyes can be used to detect apoptosis. It is also true to say that…

Cell Cycle Analysis by Flow:  DNA Stains and Beyond

Cell Cycle Analysis by Flow: DNA Stains and Beyond

While you can observe mitotic cell cycle progression using immunofluorescence, flow cytometry is a great tool to delineate details that aren’t apparent by chromosomal morphology alone. DNA stains are a great way to get a general idea of what your cells are up to. There are also a number of other stains you can use…

How to Destroy your Flow Cytometry Data in 3 Easy Steps: Snap, Crackle, and Pop

How to Destroy your Flow Cytometry Data in 3 Easy Steps: Snap, Crackle, and Pop

While many scientists are methodical and precise, some of us like to live on the edge. Read a protocol all the way through? No thanks, I’ll take my chances and guess what concentration of HCl I should use. Label my tubes with the correct content? Puh-lease – it’s much more exciting deducing which is which…

Flow Cytometric Apoptosis Assays for Cell Death
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Flow Cytometric Apoptosis Assays for Cell Death

Apoptosis, often called programmed cell death, is a carefully regulated process that is part of normal development and homeostasis. Apoptosis is morphologically and biochemically distinct from necrosis, which is conversely called accidental cell death. Dysregulation of apoptosis is implicated in disease states such as cancer, autoimmune disease and degenerative conditions. Apoptosis consists of an orderly…

Top 5 Tricks for Using FlowJo

Top 5 Tricks for Using FlowJo

Are you planning to do cellular immunology research?  Then chances are you will be introduced to the flow cytometer –  “a modern immunologist’s best friend.” This modern magic box is a highly versatile machine packed with cutting-edge fluidics and photonics (lasers). Combined with the monoclonal antibodies conjugated to fluorochromes capable of emitting light signals from a…

Basic Parameters Measured by a Flow Cytometer: What is Scattered Light and Absolute Fluorescence?

Basic Parameters Measured by a Flow Cytometer: What is Scattered Light and Absolute Fluorescence?

In a previous article, we went over the basic understanding of the inner workings of a flow cytometer. It’s important to grasp the types of measurements that are being made and, perhaps more importantly, what measurements are NOT being made. For simplicity’s sake, we’re going to frame this discussion in terms of a classical flow…

Remote Cytometry: Help from beyond!

Remote Cytometry: Help from beyond!

The idea of accessing one computer from another is long established. Unfortunately, we often have visions of hackers sneaking in and stealing our data when we have most to lose. However, this type of technology can aid us in a lot of applications and to those of us who work in cytometry the benefits are (somewhat) clear. No More ‘Fail’ Moments Many researchers know the dread of…

Sorting Large Cells and Materials by Flow Cytometry

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…

Spot the Difference: 5 Ways to Improve the Presentation of Your Flow Cytometry Data

Spot the Difference: 5 Ways to Improve the Presentation of Your Flow Cytometry Data

Take a look at the dotplot below, are you happy with the way it’s presented? Do you think that you could recreate that experiment? If you were a reviewer, would you accept that figure? Sure, it’s flow plot, it shows 3 populations of which two are gated. Read many journals and you will see data…

An Introduction to Spectral Overlap and Compensation Protocols in Flow Cytometry

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…

The Exciting (and Emitting) World of Fluorescence

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…

Catching Greatness: Measuring Cellular Degranulation

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…

Gateway to the Cellular Kingdom: Cell Fixation and Permeabilization Techniques

Gateway to the Cellular Kingdom: Cell Fixation and Permeabilization Techniques

One of the much sought after question asked by many researchers worldwide is – “What is the gene expression profile of a single cell within a heterogenous pool of cells?” While mass cytometry is the current ‘hot’ methodology for single cell analysis, the good old flow cytometry can help us perform rapid analysis of single…

Locating Your Cellular Apoptosis Squad: Annexin V Staining Assays

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…

MIFlowCyt Guidelines: Helping You to Publish Your Flow Data

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…

Troubleshooting: No Events on Your Cytometer

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…

The Nope-Nope-Nevers of Using a Flow Cytometer

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…