Written by Tim Bushnell, Ph.D
Fluorescence compensation is one of the more difficult, understandably confusing, and misunderstood aspects of flow cytometry.
Compensation is almost always necessary when more than four or five colors are measured simultaneously. As such, understanding what compensation is, why it is necessary, and what to expect when using it, are critical for generating useful and high-quality data from flow cytometry experiments.
In order to understand compensation correctly, you need to understand the answers to 3 core questions related to compensation...
1. What is the nature of fluorophores?
The necessity of compensation arises from the nature of fluorophores, the fundamental and cherished tools ...
Written by Tim Bushnell, Ph.D
Flow cytometry is a powerful technique impacting both clinical and research.
If you’re looking for a career, flow cytometry technology can take you many places. An experienced flow cytometrist can find a job in a biotechnology company, in academia, or in a clinical setting.
Rather than focusing on specific career information, I would like to highlight one career option that has been very good for me since my transition from research scientist to core manager over a decade ago.
Core facilities, or Shared Resource Laboratories (SRL) as this Cytometry A paper made popular, represent an investment by institutions in ...
Written by Tim Bushnell, Ph.D.
In the most common applications, cell sorters are utilized to purify cells on the order of about 5-30 microns in diameter, often human or murine leukocytes, or cell lines derived from liquid or solid tissue types.
Considering this typical size range, cells between 5-10 microns in diameter are typically the simplest cells to sort. That being said, the general rules of thumb in forming sample preparation, instrument setup, and sample collection are critical to generate sorts of high purity, recovery, and viability — the hallmarks of sort quality.
To this end, there are 4 steps you must take ...
Written by Tim Bushnell, Ph.D.
Do you annotate your data properly?
If you don’t, you're not alone. Most people don’t take advantage of the keywords embedded in the FCS (Flow Cytometry Standard) files to annotate their data. FlowJo makes this easy to do.
First off, what are keywords and why do you want to use keywords?
The flow cytometry standard file formats (FCS) was developed by the International Society for the Advancement of Cytometry (ISAC) to create a standard file format for all cytometers to use. The purpose of this was so that no matter who manufactured the instrument, or the analysis software, files ...
Written By Tim Bushnell, Ph.D.
Good flow cytometry depends on a high-quality, single cell suspension. If the cells put through the instrument are not of high quality, the ensuing data will be difficult to analyze.
Likewise, if the sample is clumpy, one will not be able to readily distinguish cells of interest from the clumps they are attached to. Thus, sample preparation becomes the critical first step in any flow cytometry experiment.
As Henry Ford once said, “Before everything else, getting ready is the secret of success.”
For the purposes of this article, our attention will be focused on mammalian cells and cell-lines. ...
ExCyte is the world’s leading flow cytometry training company. Whether you are a core manger looking to keep your users on the cutting edge, or a business professional looking to enter into this rapidly growing market, ExCyte can help. We have the information and experience you need.
ExCyte is currently booking beginner and advanced flow cytometry courses around the world. Click on the Calendar box above to see where we will be next.
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 ...