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5 Steps For Accurate Flow Cytometry Statistical Analysis Results

Written by Tim Bushnell, Ph.D At the end of many experiments, the question of statistical analysis rears its ugly head. When it comes up, many researchers freeze not knowing how to proceed, and they muddle through as best they can. With some proper planning and forethought, this doesn’t have to be the case. To resolve this analysis dilemma, it is important to begin thinking about the statistical analysis during the initial designing of the experiments. This is where some critical decisions need to be made to ensure that, if there are statistically significant findings to be uncovered, the data will be sufficient to ...

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3 Guidelines For Setting Compensation Controls In Flow Cytometry Experiments

Written by Tim Bushnell, Ph.D Fluorescence compensation is not possible without proper controls, so it is critical to spend the time and effort to generate high-quality controls in the preparation of an experiment. First, recall that compensation is a consequence of spectral overlap, which occurs because the fluorescence emission spectra of essentially all fluorophores are wider in wavelength range than the optical filters that we use to measure those fluorophores. Because of this, fluorophore emission will often overlap with more than one filterset on the cytometer, leading to the detection of the signal from one fluorophore by multiple detectors (i.e. spillover). The more ...

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How To Use A Threshold To Reduce Background Noise In Flow Cytometry

Written by Tim Bushnell, Ph.D On most flow cytometers, the photomultiplier tube (PMT) is the interface between the fluidics system and the electronics system. It is the PMT that converts the photons emitted from the fluorochromes into the electronic current that is digitized and ultimately converted to the value stored in the listmode file. Any stray photon of light or random electron emission from a dynode will cause a cascade, and ultimately a photocurrent. This is often known as dark current. The figure below shows the idealized idea behind this concept. Figure 1: Stylized signal coming off a PMT showing the dark current ...

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How To Set And Monitor Optimal Voltages For A Flow Cytometry Experiment

Written by Tim Bushnell, Ph.D When a researcher first sits down in front of the flow cytometer, they are faced with several choices. One of the most confusing is, “What voltage do I set my detector at?” Unless the researcher is running a fixed voltage system (Accuri, and others), this choice can dramatically impact the sensitivity of the instrument, making or breaking the experiment. In the days of the analog flow cytometers, voltages were set by placing a quadrant gate on a bivariant plot, with the lower left quadrant encompassing the first log decade. Unstained cells would be run on the instrument and ...

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Strengths And Weaknesses Of Isotype Controls In Flow Cytometry

Written by Tim Bushnell, Ph.D Controls are critical for minimizing the effects of the variables in a scientific experiment so that the effect of the independent variable can be accurately measured. In flow cytometry, there are a host of important controls necessary to properly interpret data generated in these experiments. Some of these controls include compensation, fluorescence minus one (FMO), stimulated and unstimulated, reference, and controls. When it becomes time to publish, the proper use of these controls is critical in convincing the reviewer and reader that the data has been properly analyzed. The isotype control is an experimental control where a sample ...

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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.

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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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