A colleague related the following conversation/mini-rant that happened in their facility just the other day. The names have been changed to protect the innocent.
New User #1: “Hi, I want to sort and run a 10-color panel on preparation of dissociated rat prostate tumor cells. Can you tell me what I need to order so we can do this next week?”
Wise Flow Director: Mental facepalm. “Can you be a bit more specific?”
New User #1: “I need to look at all the different cells in this prostate tumor and sort them for RNAseq. What colors do I need?”
Sadly, this scenario ...
How do you follow best practices in flow cytometry to improve reproducibility?
Reproducibility is in the science spotlight these days. With the growing body of evidence showing how much translational research is not reproducible, funding agencies and journals are taking note.
Flow cytometry, as a technique, has changed and developed over the years, with researchers constantly evolving and evaluating best practices based on technological developments.
However, in the dark recesses of old lab notebooks, there still exist the time-worn protocols of yesteryear that come back to haunt the next generation of graduate students. The lure of getting a head start by using an ...
What is doublet discrimination?
You are probably familiar with the term, “doublet discrimination” or “doublet exclusion”, and have likely included this flow cytometry measurement into at least some (if not all) of your gating strategies.
Even though you may utilize this important gating strategy, you may not have had the chance to delve deeper to explore exactly what doublets are and why it’s critical to exclude them. This article aims to do just that.
What is doublet discrimination?
The first aspect to understanding how a doublet exclusion gate works is to define what a doublet is. Most straightforwardly, a doublet is a single event ...
Signaling pathways are of great interest in many areas of research because dysregulation of these pathways can lead to diseases such as cancer and lupus. This also means these pathways contain potential therapeutic targets to help treat these diseases.
The power of flow cytometry is the ability to analyze millions of events in a short period of time. This allows for the analysis of dynamic cellular processes in a phenotypically defined manner.
The process of cellular signaling relies on changes in the phosphorylation state of proteins to either up or down regulate downstream processes (Figure 1).
Figure 1: The PI3Kinase signaling pathway has ...
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.
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 ...