How To Detect Microvesicles, Microparticles, And Ectosomes By Flow Cytometry
Written by Phil Hexley, Ph.D.
There seems to be a big fuss about small flow these days.
What is small flow?
Microvescles, microparticles, ectosomes—the terminology is all over the place but one thing is certain…
There is a need for flow cytometric analysis of extracellular vesicles.
To avoid ambiguity in this post, the population of extracellular vesicles discussed here shall be called microvesicles.
These small, membrane bound fractions of cells released during activation and apoptosis are in the 0.1 to 1 micron diameter size range (or there about depending on which article you read).
Released in vast quantities and carrying a variety of traits from the originating cell these microvesicles are known to be involved in a wide range of processed from stem cell renewal and tumor metastases through to coagulation and inflammation, just to name a few!
They key is that microvesicles have a huge potential to be important biomarkers in early disease detection and/or treatment progression.
What’s Down There? Debris Or Microvesicles?
Microvesicles have been known about for a long time, b ...Read More
5 Important Peer Review Questions To Answer Before Submitting Your Flow Cytometry Data
Publish or perish remains the mantra of science.
All the experiments and experience in the world do not count if you are unable to communicate your results to the scientific community.
As part of that communication process, your paper will undergo the dreaded ‘Peer Review’ process. If you wish your paper to survive the peer review process, you must collect, analyze, and present your data properly—before you submit your paper.
A review of the following peer review questions, as well as how to answer these questions, will help ensure your paper is not rejected.
5 Questions Peer Reviewers Will Ask
As one who is asked to peer review papers regularly, especially those with flow cytometry data, there are 5 specific questions I ask when reading a manuscript.
If these questions are not adequately answered or explained in the paper, it raises red flags and ultimately leads to rejection.
1. What is the scientific hypothesis?
It all begins with the scientific hypothesis being tested. One of the critical parts of the paper is a clear statement of the reason for the study.
While it may seem ...Read More
3 Types Of Flow Cytometry Beads That Will Help Get Your Data Published
When starting a flow cytometry experiment, you should begin with the end in mind.
What data do you need to generate to support your other data? What data would really impress the reviewers in your study section and get your lab the grant you need? What data do you need to land your first tenure-track position?
Planning with your end goal in mind is important, but don’t forget the details along the way. Your cells are important, but ensuring your experiment is well set-up and your instrument properly maintained is just as important as elegant experimental design and good writing.
The 3 Best Flow Cytometry Beads
To make certain your instrument is set up correctly for your experiments, manufacturers have developed defined polystyrene beads.
These beads’ consistent nature helps you to assess how your instrument is behaving, helps you set up proper compensation matrices, and helps you generate volumetric counts of your cell populations. The best beads of the bunch in each of these 3 areas are highlighted below…
1. Quality control beads.
Manufacturers make certain that when you ...Read More
7 Advanced Flow Cytometry Data Analysis Tips For Multi-Color Experiments
There was a time when two- and three-color experiments were considered very complex flow cytometry experiments.
A limited number of dyes and light sources meant a limited number of options overall.
In fact, the first experiments sorting cells with fluorescently-labeled antibodies were performed by Len Herzenberg in 1972 and could only detect fluorescence from an Argon laser source above 530 nm. These early experiments were performed using rhodamine- and fluorescein-tagged antibodies.
Times have changed. Now, we have spatially separated lasers and a seemingly unlimited number of dyes.
From 2-Colors To 10-Colors
Not long after Herzenberg’s work, Howard Shapiro began designing a series of multiparameter instruments. While instrumentation was evolving, there was parallel development in fluorescent colors to allow for detection of more antigens.
For example, the patent for the phycobiliprotein uses in FACS and other applications was issued to Glazer, Oi, and Stryer in 1995. Then, in the early 2000’s, Alexa Fluor dyes came onto the scene.
In today’s world, many scientists have acce ...Read More
Why Understanding The Jablonski Diagram Will Help You Publish Your Flow Cytometry Data
We are all used to interruptions during our working day, from the ping of an email notification to the knock of a fellow researcher who wants to troubleshoot their experiment.
Fortunately, most of these interruptions only last a few minutes. Some past researchers were not so lucky.
Imagine your work being interrupted by a war. Imagine it being interrupted by two wars that you had to fight in.
Alexander Jablonski often had his studies interrupted, not by emails or colleagues, but by war. Jablonski’s work was held up for years due to military service in two wars. First, he served in the war for Polish independence in 1916, then he served again in the Polish-Bolshevik war in 1920.
In 1930, after the wars were over, Jablonski earned his doctorate. His dissertation, entitled “On the influence of the change of wavelengths of excitation light on the fluorescence spectra” laid the foundation for the rest of his career in physics.
A few years later, in 1935, he created what we flow cytometrists call the Jablonski Diagram.