Flow cytometry is a numbers game. There are percentages of a population, fluorescence intensity measurements, sample averages, data normalization, and more. Many of these common calculations are useful, but surrounded by misconceptions. This primer will help you decide which calculation to use, when to use it, and how to interpret the results.
1. Staining Index
The staining index (SI) is a way to measure the relative brightness of a fluorochrome and compare it to other fluorochromes in a biologically relevant manner.
The SI is useful for ranking fluorochrome brightness on your instrument of choice. It is also a useful tool for evaluating titration data.
Written by Tim Bushnell, Ph.D
The field of medical therapeutics is moving into the area of precision medicine. In a global sense, precision medicine requires the doctor to assess a patient's unique disease state — the susceptibilities and resistances of the disease targets to the arsenal of medicines at the physician's disposal.
This is leading the push towards devising more nuanced tools, and an understanding of what specific patient characteristics dictate which tools to use.
For precision medicine to work, we must be able to identify biomarkers that are expressed on diseased cells, but absent on the normal cell.
An example of this type ...
As new instruments come on the market, vendors are quick to provide data proving the systems’ prowess including sensitivity, speed, and such. These are important characteristics of the instrument, and should be reviewed. However, the real questions that should be asked about any new instrument should look beyond these benchmarks. Specifically, the questions that often come to mind include:
Will the new instrument improve current experimental workflows?
Will the new instrument enable new and novel experimental questions?
Will the new instrument help improve the reproducibility of experiments?
Evaluating the instrument in the context of these questions will help determine if acquiring ...
Written by Tim Bushnell, Ph.D
The ultimate goal of any experiment is to analyze data and determine whether it supports or disproves a given hypothesis. To do that, scientists turn to statistics.
Statistics is a branch of mathematics dealing with the collection, analysis, interpretation, presentation, and organization of data. In applying statistics to, e.g., a scientific, industrial, or social problem, it is conventional to begin with a statistical population or a statistical model process to be studied.
One of the first important concepts to take from this definition is the idea of a population. An example population might be all the people in ...
Written by Tim Bushnell, Ph.D
One of the most common assays in flow cytometry is the surface labeling of cells with antibodies. Often termed “immunophenotyping”, it allows the researcher to identify, count, and isolate cells of interest in a mix of input cells. Every lab has their own favorite protocol, handed down from some hallowed, chemical-stained notebook, and followed as exactly as making a souffle.
The real questions are, which of those steps are critical, and (with changes in instruments and theory) what other factors should be considered when staining cells? This article will focus on staining immune cells, but the principles ...
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MedImmune purchased a site license of Expert Cytometry’s Mastery Class. We have over 100 scientists using the program and the feedback has been phenomenal. I’m amazed at how much the scientists are coming to me with questions that are much more advanced than what I used to get before having access to the Mastery Class. I’m now able to focus my attention, and that of my staff’s, to more advanced questions and inquiries specific to the researcher. We are also having less trouble with the mistakes that new users make and spending less time dealing with troubleshooting basic issues. As the core director at MedImmune, I definitely recommend the Expert Cytometry Mastery Class to all cores, particularly in a corporate setting, where we simply do not have the time to train all of the new users in best practices. My core is running 10 hours a week, ...
Expert Cytometry provided an excellent course to our group. The course was over two days. On day one, the instructors provided a great overview of core concepts of flow. On day two, we were able to customize the course to our needs. It was very valuable and I would recommend that if your group is starting to use flow more heavily, that you bring Expert Cytometry in for a course. There are nuances that I never knew and it immediately helped our group perform better experiments.
Before taking this course, my experience with flow was limited to simple 2 color experiments. Then I was asked to perform a 10-color complete immunophenotype experiment in non-lymphoid tissues. With a ton of help from a long time co-worker and friend, I was successful with the effort. She highly recommended the Excyte Mastery Course to me and I can say she was right. This course truly elevated my knowledge and understanding of flow cytometry. It was very organized, concise and covered everything from understanding your instrument, optimizing it's performance, designing multicolor panels and executing a flow cytometry experiment with all the necessary controls. I look forward to learning more from the advanced seminars and webinars. I highly recommend this course.
I was (and still am for the most part) completely ignorant of the details of flow cytometry. The Expert Cytometry team provided explanations and helped me understand the technology to the point where I was able to intelligently and efficiently manage both of our contract flow labs. Expert Cytometry provided independent assessments of the quality of the work and data from the labs that allowed me to make good decisions regarding the results. Finally, I was always amazed at how quickly Expert Cytometry responded and completed the analyses when we were on short timelines. We continue to work with ExCyte and will for the foreseeable future.
Our group at BlueBirdBio was relatively new to flow. We brought ExCyte in for a basic course, and also to help train us on best practices for our new flow cytometer. The level of flow cytometry knowledge ExCyte brought with them was amazing. I can’t believe how much experience ExCyte has and how much they bring to the courses they provide. ExCyte was able to answer all questions with real world examples. The course brought the theory of flow to the real world and helped us understand the concepts more easily. ExCyte was professional, courteous, and it almost felt like they became part of our group while we were working together. I know I can ask ExCyte any questions whenever I need them and I hope to continue to work with them as we move forward. I have no doubt we’ll bring ExCyte back for another course in ...