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Ensuring Reproducibility In Flow Cytometry

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Reproducibility In Flow Cytometry Requires Correct Compensation

Why do we have to compensate flow cytometry data? Newcomers to flow cytometry are often confronted with one of the most confounding issues in flow cytometry. That is, trying to understand the whole idea of “compensation”. It can be explained theoretically, mathematically, by trial and error, or by “take my word for it”. Depending on the audience, a combination of these are used to get the point across. Simply put, compensation is the mathematical process of correcting the spectral spillover of a fluorochrome into a secondary detector. It relates to the physics of fluorescence. To understand what this means, let's start with ...

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Best Practices In Flow Cytometry Compensation Methodologies

What is compensation and when should you do it? In this first of 3 blog articles, we will discuss the principles of compensation, as well as why it is important, and how to perform compensation. Subsequent articles will discuss the rules that must be followed for proper compensation and some of the common compensation myths that permeate the field. It all begins with an understanding of the process of fluorescence. After excitation, a fluorescent molecule emits a photon. This photon has an emission maximum — that is, the most probable photon wavelength that will be emitted. However, this emission is not so ...

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The Need For Speed In Flow Cytometry Data Analysis

Speed is a highly touted metric in flow cytometry. Look at any vendor’s website and you will see the highlights on how many events per second their instrument can acquire, how many cells can be sorted per second, and more. The limitations are imposed by the physics of flow cytometry, the speed of pulse processing, and more. With cell sorters, Poisson statistics dominate the speed calculation. As has been discussed before, the optimal sort rate is ¼ the frequency of droplet generation. Sorting faster will impact purity of the final product. One of the trends in flow cytometry is pushing the ...

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Instrument Quality Control For Reproducible Flow Cytometry Experiments

The NIH has released a series of reproducibility guidelines that scientists must address. These guidelines have been introduced because there's a lot of data showing that reproducibility in science is frustratingly lacking in certain experiments. Non-reproducible experiments waste time, money, and resources. As Begley and Ioannidis cite in their 2015 article : “The estimates for irreproducibility based on these empirical observations range from 75% to 90%. These estimates fit remarkably well with estimates of 85% for the proportion of biomedical research that is wasted at-large.” Reproducibility is a mindset, and involves an overall analysis of the scientific process to identify the areas that ...

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Experimental Controls For Reproducible Flow Cytometry Measurements

With the increased focus on reproducibility of scientific data, it is important to look at how data is interpreted. To assist in data interpretation, the scientific method requires that controls are built into the experimental workflow. These controls are essential to minimize the effects of variables in the experiment so that changes caused by the independent variable can be properly elucidated. In fact, one of Begley’s 6 rules, as described by Bruce Booth, asks if the positive and negative controls were both shown. What types of controls should be considered when designing a flow cytometry experiment? Focus controls to minimize confounding variability. ...

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Testimonials
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, ...

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Johnna Wesley, Ph.D.Senior Manager, Novo Nordisk
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.   

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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.

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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.

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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 ...

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