Adaptive Biotechnologies

Getting Started with NGS Bioinformatics

Bioinformatics and NGS go together like peanut butter and jelly. But if you’re just starting out with these techniques it can be daunting.
Tips for Successful Bone Marrow Isolation

During my first year of graduate school, I learned how to isolate bone marrow. I remember watching my mentor in awe, wondering how would I be able to do such a difficult technique. Flash forward to a few weeks later and I was confidently undertaking bone marrow isolation. Learning a new technique is always daunting,…
Should You Switch from Wet to Dried Blood Samples?

A Spot of History Most of the biomedical methods used started as a curiosity. Then the one-off gains a limited use, the technology then progresses until its use becomes widespread. Just think about the arch from the curious polished glass spheres, used by Antony Levnhook to look at animalcules, to modern microscopes. The same story…
Isolating Monocytes from Whole Blood: A Step-by-Step Guide

If you look at the composition of peripheral blood, using hematology microscopy, you’ll see that it’s composed of multiple different cell types, including monocytes. It’s possible to isolate these different components to study and experiment on them directly. So, if you’ve done a few experiments and had fun with THP-1 cells, you can move on…
Thinking Outside the Box: Microscopy for Immunologists

When you think of an immunologist, you will likely imagine someone who studies the immune system… or maybe a person who speaks in a completely different language (CD? IL? The list goes on.). You may also think of a slew of assays that almost exclusively “belong” to immunologists, including ELISA, ELISpot, Flow Cytometry, chromium release…
An Introduction to Chimeric Antigen Receptors (CARs)

You may have heard about a breakthrough cancer therapy that engineers patient’s immune cells to fight their cancer using chimeric antigen receptor (CAR)-T cells. If you don’t live in the world of immunology, you may not know what a CAR is, or what it is used for. Here you’ll find a brief guide to CARs,…
How 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 set up, 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…
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…
How 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.…
The Good, the Bad and the Expensive of Whole Genome Sequencing

Whole Genome Sequencing (WGS) is still very cutting edge, sequencing technology and while there are a lot of perks to using it, there are also a few drawbacks. The good, the bad and the pricey are outlined below to help you navigate when it’s worth using WGS! Whole Genome Sequencing: The Good Lots of Data…
Post-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…
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

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…
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…
The 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…
How to Perform DNA Extraction from Dried Blood Spots Using Chelex Resin

Every bio- scientist who wants to analyze DNA knows that the process begins with the extraction of DNA from cells of interest. These cells could be RBCs, parasites, or bacteria to name a few. Furthermore, there are various DNA extraction methods1 to choose from depending on sample type, downstream analysis, and so forth. Many scientists…
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…
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…
Chromium Release Assay: The Old School Way of Testing Cytotoxic T Cells

There are several methods you can use to see if your T cells are cytotoxic, but a chromium release assay using radioactive 51chromium (51Cr) is one of the oldest. It gives good results, and is great for labs that can’t afford or don’t have flow cytometry readily available. Here, I will outline a simple method…
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…
Pre-Analytical Sample Handling: What Can You Do Better?

If you study human disease, you will likely handle a pre-analytical sample or two (or hundreds). For example, you could handle whole blood, serum or plasma, tissue biopsies, urine, fecal samples, cerebrospinal fluid, or synovial fluid—to name a few. You will probably use these samples to look for specific metabolites, proteins, or nucleic acids that provide…
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…
Four Tips for Working with Human Clinical Samples

While using human clinical samples in your research can provide robust and heterogeneous results applicable to larger portions of the population, working with these samples presents its own set of challenges. Here are some tricks I have learned to help isolate and grow your cells of interest while eliminating stromal, blood, or other undesired contaminants.…
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

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

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

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…
How to Choose Your Method for DNA Extraction from Whole Blood

Over the last few decades, PCR, next generation sequencing and microarray technologies have taken blood-based research to a new level. Modern blood-based application range from DNA fingerprinting, whole genome sequencing, blood banking to liquid biopsy and many more. Regardless of the application, pure, intact, double stranded and highly concentrated DNA extraction from whole blood is…
A Biologist’s Guide to Choosing Your Fluorophore Palette

Selecting fluorophores can be a tricky business, but we’ve got you covered in this handy how-to guide.
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…
The Care and Keeping of Primary Murine B Cells

So you want to work with mouse B cells? Primary murine B cells are a difficult, yet fascinating system to work with and can help deepen your understanding of an immunological system. You can study many things with primary B cells, including: immune activation antibody production cell-cell interactions between immune cells and immune phenotype These…
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…
A Simple Way to Measure T cell Killing Activity In Vivo

Have you ever wondered what a cell’s life is like? Do they constantly communicate to each other or do they just go on their own daily business? There is an easier way and it involves super advanced molecular “crayon” technology.
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…
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…
Five Simple Steps For a Successful MTS Assay!

The MTS cell viability assay is one of the most important yet often daunting assays to perform for researchers in cancer biology, immunology, drug delivery pharmacy, etc. This chromogenic assay is extremely dependable for assessing the effects of a drug on different cell lines. However, it is only an easy assay to master if you…
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…
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 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…
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…
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…
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…
Decoding the Genome: Applications of DNA Sequencing

The age of sequencing is undoubtedly upon us. From improving cancer diagnostics to pinning down elephant poaching hotspots, DNA sequencing is revolutionizing the world around us from the ground up. The latest video from Thermo Fisher Scientific’s “Behind the Bench” blog, 10 moments in DNA sequencing gives fascinating insights into the amazing advances being made…
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),…
A Protein Biochemist’s Bag of Tricks

If you were to peek into a protein biochemist’s bag of tricks, what would you find? A mortar and pestle for collecting samples, some columns for isolating proteins and a mass spec instrument? Perhaps. But what about those little eppendorf tubes full of enzymes and helpful molecules? Certainly, each scientist has his/her own favorite. Here…
Choosing The Right Blood Collection Tubes

So you’re designing a project using human blood samples. Maybe you’re studying blood cells using hematology microscopy, maybe you’re after genetic material or circulating biomarkers. For many basic scientists, the process of getting that blood out of a person and into your project can be intimidating. Where Do You Start? Whether you’re collecting your samples…
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…
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…
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 –…
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…
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…
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…
Six Ways to Measure T Cell Responses

T cells can be problematic to characterise because they have a wide variety of subtypes and because of the technical difficulties of studying the membrane-bound T cell receptor, but there are situations where you want to be able to do this such as analysing the degree to which immunological memory has been induced to measuring…
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…
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…
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…
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…
Which Cytokine Will I Get? How to Stimulate Human Cytokine-Producing Cells

Cells are like people: depending on their current environment, past experiences and their genetic make-up they will react differently. Treat cells in different ways, and they will produce different cytokines. There are a lot of protocols out there for stimulating cells. Depending on the species of cells you plan to stimulate, different protocols are available…
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…
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…
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…
All for one and one for all – Fluorescence Minus One Controls

Ever done a multicolour flow cytometry experiment, run all your controls, done your compensation and then started to analyse your data and realised that you can’t work out where to put your gates? To err is human… When acquiring data on a cytometer, there can be measurement errors due to counting statistics, errors in processing…
Take Control of Your World – Five Controls for Flow Cytometry

No, this is not a call for Geeks to take over the world – just a tiny part of it – the part that ensures success in all experiments or at least a good way to analyze them if they fail. There are all too many entry points for error and variability that can be…
Intracellular Cytokine Staining: Letting It All Build Up Inside

Cytokines, those small proteins that modulate immune cell responses, once translated are normally secreted rapidly out of the cell. So, previously we could only check the levels of cytokines secreted in the supernatant, but we wouldn’t know which cell was producing which cytokine. But what if we had a way to keep the cytokines inside the cell? Then we…
Biosafety in Flow Cytometry – To Be or Not to Be…

Biosafety is one of those things many scientists don’t take seriously. I would guess, that like politics, there are 40% who believe biosafety is ‘over-emphasized’ and 40% who swear by biosafety. 20% are undecided. Needless to say, I’m on the side of biosafety. And here’s why: “CDC announced today that approximately 75 Atlanta-based staff are…
Sorting Single Cells – What Do You Need to Consider?

Flow cytometer and cell sorter manufacturers have invested considerable resources to design instruments that are the “fastest in the ‘hood” either in terms of cells analyzed per second, or in total throughput. The general idea is the faster you can go, the quicker you can identify rare cells, and produce sorted populations containing large numbers…
10 Useful Tips For Improving Your Sorting Experiments

After a very naïve start in flow cytometry thinking that published protocols will work without fault – needless to say, that did not work out in my favor – I realize now that following a few simple steps can go a long way, and will undoubtedly save you time in the end. So, here are…
An Introduction to Gating in Flow Cytometry

What is one of the first things you do when you sit down at the flow cytometer and start looking at your cells? You start drawing polygons and setting gates. To the neophyte the gating process can look a little random – why do you exclude those dots but not these? But gating in flow…
How to keep your Flow Cytometry Core Facility Happy

The helpful people who work in your flow core are an amazing resource of information for your experiments and are the people with the power to get experiments done. However, you need to be nice to these powerful people, so here are a few handy hints: Its all down to the timing When booking machines,…
Seeing is Believing: An Introduction to Imaging Flow Cytometry

What if there was a way to take the power and speed of a flow cytometer and couple it with the resolution of a microscope? Imaging flow cytometry does just that! Flow cytometry is a very powerful tool for the complex characterization of cells and cell populations but flow cytometers can also be thought of…
Getting in the Flow: Online Resources for Flow Cytometry

You are sitting in a seminar when you glimpse it: the figure that beats all other figures – a beautiful contour plot – and you realise that a similar figure is what you need to publish your paper in a high-ranking journal. You know the basics of flow cytometry, but admit you need a little…
Western Blot, ELISA, SPR, Biosensor Assay or PCR: Which Technique Should I Use?

Stimulation of cells/tissue with a given stimulus (e.g., a cytokine) is a common experimental setup in any cell biology lab. The cellular response to the external stimulus e.g., the activation/deactivation of intracellular signaling pathways and/or the secretion of proteins is often the research goal, and there are a number of different methods that you can use to analyze such…
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),…
Evolution Flow: The Historical Background of Flow Cytometry

For those of you who print out the results that you have acquired from the Flow Cytometer to stick in your lab book, did you know that inkjet printers and cytometers have a shared history? Flow Cytometry is less than 50 years old and machines today still use some of the same principles as the…
Garbage in, Garbage out? Quality Control of Your NGS Data

So, you’ve just received a call from the core facility that you hired to prepare and sequence your libraries. The facility director tells you that the sequence data from your next generation sequencing (NGS) experiment does not look good. You panic and, perhaps, let loose a scream of frustration—aaarrrrggghhhh! This project was going to be…
It’s Like Getting RNA From a Blood Sample

So you have some blood stored in the -20C or -80C and you want to isolate RNA from the samples. If you wanted DNA, you would have many products to choose from. But for RNA, your choices are more limited. Obtaining RNA From Frozen Blood is Difficult Why is that? The reason is that most RNA from…