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If You Don't Know This About GFP, FITC, And PE, You Might Publish False Flow Cytometry Data

If You Don't Know This About GFP, FITC, And PE, You Might Publish False Flow Cytometry Data

By: Tim Bushnell, PhD

When we learn about fluorescence, the first thing we are told is that fluorophores emit photons that are higher wavelength than the photons that they absorb. What this specifically refers to is the stokes shift, which results from non-radiative energy transfer during the fluorescence process. When a photon is absorbed by a fluorophore molecule, some of the resultant energy is lost in molecular vibration and movement (among other things) so that the energy released after fluorescence is lower than the energy absorbed. Since wavelength is inversely proportional to energy, this lower output energy light is higher in wavelength than the…

How To Do Phospho-Flow Cytometry

How To Do Phospho-Flow Cytometry

By: Tim Bushnell, PhD

I often have researchers come into the core wanting to look at the activation and downstream signaling events that occur in different immune cells. These events occur in response to signals such as cytokines, chemokines, various receptor ligands, and the engagement of the T cell or B cell receptors. The signaling events are also characterized by the initiation of several phosphorylation events. Measuring Phosphorylation Events When this is the case, I recommend that the researchers set up a phospho-specific flow cytometry, or phospho-flow, experiment. These types of experiments measure the phosphorylation state of intracellular proteins at the single cell level.…

5 Mistakes Scientists Make When Doing Flow Cytometry Proliferation Experiments

5 Mistakes Scientists Make When Doing Flow Cytometry Proliferation Experiments

By: Tim Bushnell, PhD

Measuring cell proliferation can be done in a number of ways. There is the below tried and true method of counting cells. This straightforward assay can help determine if the cells are proliferating and by comparing counts. Here, a researcher can determine that the experimental treatment is increasing cell growth. A second method of measuring proliferation involves using a radioactive tracer like 3H-Thymidine. In this assay, the amount of the isotope taken up by the cells correlates to the amount of DNA synthesis, and therefore growth. Of course this requires using radioactivity and all that entails. A third method is…

How To Design Accurate & Effective Flow Antibody Panels (or, What's An OMIP?)

How To Design Accurate & Effective Flow Antibody Panels (or, What's An OMIP?)

By: Tim Bushnell, PhD

I was at a meeting talking about the principles of panel design. At the end of my talk, I had an investigator approach me and ask why he was not making progress on his 15-color panel that he started developing. So, I asked how long he’d been working on it. A month. That was his response. This might shock some of you but a month is not very long when it comes to designing an accurate and effective antibody panel for a flow cytometry experiment. Multicolor panel design requires a delicate balance of biology and physics. Understanding the biology of…

10 FlowJo Version X Hacks That Will Help You Publish Your Flow Cytometry Data

10 FlowJo Version X Hacks That Will Help You Publish Your Flow Cytometry Data

By: Tim Bushnell, PhD

So you just got the most amazing results of your life and you can wait to show it off in lab meeting, or create the figure for a publication. Here are a couple of tips that will help you ensure that everyone else also sees how amazing your data is! 1. If you only have a few events, use the option to “show large dots”. When you only have 4-5 events in a population, it can often be difficult to see. If you turn off the high resolution, you can see the data better. Double click on a plot in…

Top 10 Flow Cytometry Resources

Top 10 Flow Cytometry Resources

By: Tim Bushnell, PhD

I’ve been in the world of flow cytometry and cell sorting for a very long time. Now, don’t worry, this isn’t going to be some lament about the “good old days.” Well, maybe just a little. But there will be helpful takeaways, I promise. I was trained by the incredible staff in the core facility at the University of Arizona. When I moved to UC Davis and proceeded to start up a core facility, there were very few resources to guide me. No Google communities and LinkedIn groups. Email was new, there was the Purdue Cytometry List, but that was…

4 Fluidics Tips That Will Change Your Flow Data For The Better

4 Fluidics Tips That Will Change Your Flow Data For The Better

By: Tim Bushnell, PhD

Friday is the 4th of July in the US – and we celebrate that day with picnics, spending time OUTSIDE the lab and fireworks. And our Independence, but I’m not up for a political discussion right now. For flow geeks, fireworks are like flow cytometry – they happen in the dark, they are full of many bright colors, and we’re all looking for the patterns the colors make. So in honor of a day outside the lab, it seemed appropriate to talk about fluidics… going with the flow for best results. A flow cytometer has three major components – fluidics,…

Counting Cells Will Save Your Flow Cytometry Experiment, If You Do It Like This

Counting Cells Will Save Your Flow Cytometry Experiment, If You Do It Like This

By: Tim Bushnell, PhD

Fast, accurate or efficient – pick two. How to decide when you can’t have it all. The hemocytometer is considered the gold standard for cell counting. Invented by Louis Charles Malassez, this precision etched microscope slide can allow the researcher to count their cells under the microscope with amazing accuracy. It is inexpensive, relative to other methods, but is by no means the most efficient or fast method out there. The single biggest key to using a hemocytometer is training, training and more training. Since the investigator is visually inspecting the cells within a boundary, the rules of what cells…

Top Flow Cytometer

Top Flow Cytometer

By: Tim Bushnell, PhD

What is the top flow cytometer? The easy answer is the flow cytometer that matches your needs and fits within your budget. However, before running off to spend cash, consider the following. What are the current needs of the users? Evaluating the user’s needs will help define the parameters needed for the flow cytometer. This will include what excitation sources should be available, how many detectors are needed, including specialized detectors (small particle detectors), as the need for automatic sampling. All these factors will in weigh in on the What are the projected future needs of the users? This is…

Flow Cytometry Education

Flow Cytometry Education

By: Tim Bushnell, PhD

As with all complex technology there are many different levels of education that users should avail themselves of. Basic instrument operation This level of education is akin to learning how to drive a car.  At this level, the focus will be on how to put the sample on the instrument, how to adjust voltage and collect data.  Specific policies of the facility should also be covered. Advanced instrument operation At this level of education, the focus should be on advanced techniques on the instrument.  This would cover basic troubleshooting – identifying problems and how to solve them.  Advanced training should…

Proliferation Experiments

Proliferation Experiments

By: Tim Bushnell, PhD

Cell proliferation can readily be measured by flow cytometry.  Depending on the research question, there are several different techniques that can be used. Cell counting experiments This relatively straightforward experiment where the investigator uses one of several counting techniques to see if there is an increase in the populations. This can be done using a microscope and a hemacytometer, a flow cytometer with counting beads, image based tools (like the T-20, the Countess, or the Cellometer), or coulter counter type instruments (including the Coulter Counter the Scepter and the Casy counter) Cell cycle experiments One of the earliest techniques developed…

How To Do Flow Cytometry?

How To Do Flow Cytometry?

By: Tim Bushnell, PhD

Flow cytometry is a powerful tool for asking and answering questions at the whole cell level. The first step in any flow cytometry experiment is to define the hypothesis or biological question that is to be answered.  This helps ensure that flow is the correct technique for answering the question.   One major determination that needs to made is will the cells be purified (via sorting) or is the experiment just analytical. The second step in any flow cytometry experiment is to identify the reagents needed for the experiment.  In immunophenotyping experiments, this involved knowing what subpopulations that need to be…

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