4 – Experimental Design

What photon from yonder fluorochrome breaks?  It is … umm… hmmm. Let me see. Excitation off a 561 nm laser, with an emission maximum of 692 nm. I’m sure if Shakespeare was a flow cytometrist, he might have written that very scene. But the play is lost in time. However, since the protagonist had difficulty determining what fluorochrome was emitting photons, let’s consider how this could be figured out. In my opinion, one of the handiest flow cytometry tools is the spectral viewer. This tool helps visualize the excitation and emission profile of different fluorochromes, as well as allowing you…

Reproducibility has been an ongoing, and important, concept in the sciences for years.  In the area of biomedical research, the alarm was sounded by several papers published in the early 2010’s.  Authors like Begley and Ellis, Prinz and coworkers, and Vasilevsky and colleagues, among others reported an alarming trend in the reproducibility of pre-clinical data.  These reports indicated between 50% to almost 90% of published pre-clinical data were not reproducible.  This was further highlighted in the article by Freedman and coworkers, who tried to identify and quantify the different sources of error that could be causing this crisis.  Figure 1,…

I want to thank all of you who send us your questions about flow cytometry, so I thought I would dip into the old email bag and answer a few of the common ones here.  If your question isn’t answered this time, look for it to be answered in a future blog post.  Of course, if you want us to cover a specific topic, drop us a line.  1. How Fast Can I Go? This is  a common question. The allure of the ‘hi’ button is hard to resist.  The faster you go, the sooner you are finished with data…

My first introduction to flow cytometry was talking to a professor who’d brought one on a research cruise to study phytoplankton. It was only later that I was introduced to the marvelous world that’s been my career for over 20 years.   In that time, I’ve had the opportunity to work with researchers in many different areas, exposing me to a wide variety of cell types and more important assays. What continues to amaze me is the number of different parameters we can measure, not just the number of fluorochromes, but the information we can extract from samples – animal, vegetable…

Numbers are all around us.  My personal favorite is ≅1.618 aka ɸ aka ‘the golden ratio’.  It’s found throughout history, where it has influenced architects and artists. We see it in nature, in plants, and it is used in movies to frame shots. It can be approximated by the Fibonacci sequence (another math favorite of mine). However, I have not worked out how to apply this to flow cytometry.  That doesn’t mean numbers aren’t important in flow cytometry. They are central to everything we do, and in this blog, I’m going to flit around numbers-based questions that I have received…

Developments such as the recent upgrade to the Cytobank analysis platform and the creation of new packages such as Immunocluster are reducing the computational expertise needed to work with high-dimensional flow cytometry datasets. Whether you are a researcher in academia, industry, or government, you may want to take advantage of the reduced barrier to entry to apply high-dimensional flow cytometry in your work. However, you’ll need the right experimental design to access the new transformative insights available through these approaches and avoid wasting the considerable time and money required for performing them. As with all experiments, a good design begins…

Fluorochrome, antibodies and detectors are important. The journey of a thousand cells starts with a good fluorescent panel. The polychromatic panel is the combination of antibodies and fluorochromes. These will be used during the experiment to answer the biological question of interest. When you only need a few targets, the creation of the panel is relatively straightforward. It’s only when you start to get into more complex panels with multiple fluorochromes that overlap in excitation and emission gets more interesting.  FLUOROCHROMES Both full spectrum and traditional fluorescent flow cytometry rely on measuring the emission of the fluorochromes that are attached…

Here we are, at the end of an eventful year 2021. But with the promise of a new year 2022 to come. It has been a long year, filled with ups and downs. It is always good to reflect on the past year as we move to the future.  In Memoriam Sir Isaac Newton wrote “If I have seen further, it is by standing upon the shoulders of giants.” In the past year, we have lost some giants of our field including Zbigniew Darzynkiwicz, who contributed much in the areas of cell cycle analysis and apoptosis. Howard Shapiro, known for…

It is no secret that I am a very big fan of the Star Trek franchise. There are many good episodes and lessons explored in the 813+ episodes, 12 movies (and counting). Don’t worry, this blog is not going to review all 813, or even 5 of them. Instead, some of the lessons I have taken away from the show that have applicability to science and flow cytometry.  “Darmok and Jalad at Tanagra.”  (ST:TNG season 5, episode 2) This is probably one of my favorite episodes, which involves Picard and an alien trying to establish a common ground and learn…

Sun Tzu was a Chinese general and philosopher. His most famous writing is ‘The Art of War’, and has been studied by generals and CEOs, to glean ideas and strategies to help their missions. I was recently rereading this work and thought to myself if any of Sun Tzu’s lessons could apply to flow cytometry.  So I have identified 5 points that I think lend themselves to thinking about flow cytometry.  “Quickness is the essence of the war.” In flow cytometry, speed is of the essence. The longer the cells are out of their natural environment, the less happy they…

Mastering foundational concepts are imperative for successfully using any technique or system.  Robert Heinlein introduced the term ‘Grok’  in his novel Stranger in a Strange Land. Ever since then it has made its way into popular culture. To Grok something is to understand it intuitively, fully. As a cytometrist, there are several key concepts that you must grok to be successful in your career. These foundational concepts are the key tools that we use day in and day out to identify and characterize our cells of interest.  Cells Flow cytometry measures biological processes at the whole cell level. To do…

Developing variant calling and analysis pipelines for NGS sequenced data have become a norm in clinical labs. These pipelines include a strategic integration of several tools and techniques to identify molecular and structural variants. That eventually helps in the apt variant annotation and interpretation. This blog will delve into the concepts and intricacies of developing a “variant calling” pipeline using GATK. “Variant calling” can also be performed using tools other than GATK, such as FREEBAYES and SAMTOOLS.  In this blog, I will walk you through variant calling methods on Illumina germline RNASeq data. In the steps, wherever required, I will…