3 Ways to Use Flow Cytometry for Your Activation Experiment
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3 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,…

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…

Sheath Pressure: Nozzle Size Does Matter

Sheath 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…

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…

The Difference Between an Image, Flow, Time-lapse and Cell-sorting Cytometer

The 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…

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…

The History and Future of Fluorescent Labels: We’ve Come a Long Way, Baby!

The 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…

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…

Analyzing Cell Signaling with Flow Cytometry: Go with the Flow

Analyzing 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…

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…

Ensure Reproducibility: Control for Lot-to-Lot Variation of Antibodies
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Ensure 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…

Introducing CyTOF:  Cytometry of the Masses

Introducing 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…

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…

Cell Cycle Analysis by Flow Cytometry: Flowing your Way through Life’s Cycle

Cell 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…

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…