Flow Cytometry
Nail Your Unmixing for Full Spectrum Flow Cytometry: 7 Top Tips
Spectral unmixing in flow cytometry is the key to great data from your full spectrum flow cytometry. Get this wrong, and you risk unreliable results. Read our top 7 tips from a flow cytometry core facilities expert to nail your unmixing.
Read MoreFlow Cytometry Gone Viral: Introducing Flow Virometry
Do you want to know about a cool way to detect and tell the difference between virus particles? Then read on to discover flow virometry!
Read MoreThe CS&T Report: Its Troubles, and How to Fix Them
What is one of the first steps of the flow cytometer start-up routine? To check if the cytometer is in good shape, of course! Everyone wants to run on a reliable instrument, and assessing the daily performance of the instrument show us its behaviour over time and allows us to react fast in case of…
Read MoreAn Introduction to Alexa Dyes
Long before “Alexa” was a household name, Alexa dyes were an established series of fluorescent dyes. The inventor Richard Paul Haugland named the dyes after his son Alex. Originally a trademark of Molecular Probes, the Alexa family is now a part of Thermo Fisher Scientific. Alexa dyes are frequently used as labels in fluorescence microscopy,…
Read More3 Ways to Use Flow Cytometry for Your Activation Experiment
Studying immune cell activation allows scientists to understand the way the body mounts a response to a specific infection, autoimmune diseases, or cancer. This knowledge plays a direct role in developing more efficacious vaccines and therapies. When tasked with capturing information on immune cell activation, flow cytometry remains the gold standard due to its versatility,…
Read MoreHow Social Media Can up Your Flow Cytometry Game
Flow cytometry is an ever-evolving field, and new technologies and innovations seem to emerge every week. So how is a scientist supposed to keep abreast of so much information, and weed through all of the unnecessary information at our fingertips? Try social media! Yes, I went there. Social media is everywhere. You can use it…
Read MoreHow to Unclog Your Flow Cytometer
Welcome back, fellow flow cytometry friend! I am sure that you are rocking your data acquisition at this point, having perfected your understanding of panel setup, fluorophore usage, and using the flow cytometer of your choice. However, with as many samples as you are running, it is possible that you may be experiencing a little…
Read MoreFive 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…
Read MoreHow to Maintain Healthy Interactions with Flow Cytometry Personnel
No matter the ingenuity of your science or the capabilities of your core facility, your results are only as good as your peer-to-peer relationship with the flow cytometry personnel. The following points are from my experience as a flow cytometry facility manager, but they are equally applicable during discussions with managers from any core area.…
Read MorePost-sorting Checks and Measures
In my previous article I discussed steps you can implement to ensure that a sample is ready for cell sorting. But now it’s time to make sure the sort worked. Here are a few sorting checks and measures to ensure that all’s well that ends well. Post-sorting Checks and Measures Re-evaluate Your Catch Tubes Sorting…
Read MoreGuidelines 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…
Read MoreSheath Pressure: Nozzle Size Does Matter
Hello again, fellow Flow Cytometry Fan! It looks like you have your experiment all planned out, including staining protocols and gating schemes, and are ready to get some paradigm-shifting data. But before we start “plugging-and-chugging” samples through your cytometer of choice, we need to make sure that the nozzle size and sheath pressure are set…
Read MoreThe 3 Most Common Flow Cytometry Fallacies
Flow cytometry is fast evolving from a method only revered by immunologists, to one used by nearly every biological specialty. It’s pretty much my favorite tool. Unfortunately, as with most lab techniques, much of flow cytometry is taught on the job without a lot of standards. And too often bad habits are passed along like…
Read MoreChromosome 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…
Read MoreHow 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…
Read MoreAre 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…
Read MoreThe Difference Between an Image, Flow, Time-lapse and Cell-sorting Cytometer
Ah, cell counting — it’s the oldest trick in the book! Well, not really, but people have been developing methods for counting cells since the late 1800s. It has been around for a while. But what different methodologies are available to biologists now? Well, hold on, because you’re in for a treat! In this article, we…
Read MoreHierarchical 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…
Read MoreHow 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…
Read MoreHydrodynamic Focusing in Flow Cytometry
If you have sorted samples or phenotyped cells by surface expression of proteins, you’ve probably wondered how each cell is sorted or phenotyped in a flow cytometer? This question seems trivial, but in reality it took a while for engineers to figure it out. Before I get into today’s topic on “hydrodynamic focusing,” I’ll walk…
Read MoreMultiplex 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…
Read MoreDemystifying 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…
Read MoreThe History and Future of Fluorescent Labels: We’ve Come a Long Way, Baby!
If you’ve been keeping up with our recent series of articles, welcome back! If not, you can catch up on how fluorescence works or what not to do with your flow experiment. In short, we have been discussing fluorescent labels and their role in flow cytometry. Today, I’ll round out our discussion by touching on…
Read MoreCorralling 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.…
Read MoreLighting 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…
Read MoreAnalyzing Cell Signaling with Flow Cytometry: Go with the Flow
Phosphorylation Equals Cell Signaling! How do cells communicate and respond to their environmental cues? This question has been on the hot list for scientists ever since the discovery of the cell. Cells use signaling cascades based on biochemical reactions to deliver or receive messages. How cool is that? The major secret of cell signaling was…
Read MoreDetection 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…
Read MoreCell 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…
Read MoreHow 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…
Read MoreFlow 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…
Read MoreTop 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…
Read MoreEnsure Reproducibility: Control for Lot-to-Lot Variation of Antibodies
When starting a long-term experiment, you need to take a lot of things into consideration (availability of cells, reagents, planning time points), but do you ever think about your antibodies? If you buy an antibody from a manufacturer, run out half way through the study, and buy the same antibody again, have you thought about…
Read MoreIntroducing CyTOF: Cytometry of the Masses
Flow cytometry remains unparalleled as a single-cell analysis technology. The ability to analyze 14 or more fluorescent parameters on a million cells or more allows for detailed understanding of complex biological processes. The Problem With Traditional Flow Cytometry One limitation of flow cytometry is the reliance on fluorescent tags. Even with careful panel design, loss…
Read MoreMass(ively powerful) Cytometry
Mass Cytometry is a relatively new technology which has recently featured in many high-impact journals. You may have read about instruments including the CyTOF, CyTOF2, and more recently, the Helios. With these instruments becoming more widespread, you might find yourself asking, what is mass cytometry, and what can it do for you? The Basis: Conventional…
Read MoreLocating 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…
Read MoreMIFlowCyt 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…
Read MoreTroubleshooting: 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…
Read MoreThe 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…
Read MoreBasic 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…
Read MoreCell Cycle Analysis by Flow Cytometry: Flowing your Way through Life’s Cycle
Over the past few decades the mammalian cell cycle has been well documented. Although there are lots of checkpoints as cells move through the cycle, we can very simply divide the cell cycle into three stages according to the DNA content in the nucleus. When cells are either quiescent or not dividing they have the…
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