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3 Considerations To Ensure Your Cell Sorting Flow Cytometry Experiments Run Smoothly

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Written By: Tim Bushnell, Ph.D.

Cell sorting is such an important technique because it’s a gateway tool for many other downstream applications.

Assays such as culturing cells, genomic analysis, proteomics, injections into mice and the like are enabled by cell sorting, allowing researchers to use a homogeneously defined population of cells in their experiments.

With single-cell genomics we can readily characterize a population in great detail, but we have to have a single purified population for it to work.

That’s where the cell sorter comes in.

There are all sorts of applications we can do with a purified population and with the advances in sorting and fluorescent technologies, it is possible to isolate complex phenotypes from rare populations.

Fluorescent proteins are a great example of being able to look at the expression of the inter se or the marker coupled to a fluorescent protein so we can sort.

We can do two or three or four of these GFP, YFP and some of the fruit fluorochrome so you can look at the expression patterns of two or three different proteins at the sam ...

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3 Questions You Should Be Asking About Flow Cytometry Controls For Your Experiments

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Written By: Tim Bushnell, Ph.D.

What are the controls that you should be considering when planning and designing your flow cytometry experiments?

Are you using isotype controls? Should you be?

Isotype controls are one of the controls used in flow cytometry today and is very controversial. In fact, I encourage people to not use it.

Are you following the rules of compensation and including the appropriate controls?

Compensation is such an important thing to get correct when running your experiments, especially if you’re doing polychromatic flow cytometry, which many of us are doing nowadays.

What types of quality controls should you have in place? Quality control is an important part of the whole experimental process any yet many people don’t worry about it or even think about it.

But if left unchecked, poor quality control can cause major problems.

For example, a colleague of mine was searching for a biomarker for a disease that they were studying. They had done several years worth of analysis and over time acquired many samples. When they went to analyze the data they fo ...

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3 Ways To Improve Flow Cytometry Troubleshooting

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Written by: Tim Bushnell, Ph.D.

I spend a lot of time working with my colleagues and clients retrospectively troubleshooting their data. This involves trying to understand and explain what might’ve happened during the data acquisition that led to the results in question.

During these discussions, I give them tips and strategies to improve their acquisition and data quality over time because we want to make sure that people have the highest quality data they can. This means that the researchers should understand and follow the best practices and are taking the time to make sure that their data is collected correctly.

There are three main areas that you need to think about when troubleshooting your experiments…

1. What do you before you start collecting data?

When you sit down at the cytometer, what are the steps that you go through to make sure the cytometer is ready?

Do you check the quality control logs?

Do you ask the operators for the quality control to see how the machine’s behaving since the last time you ran the system?

Or, better yet, do you have some sort o ...

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Using Begley’s Rules To Improve Reproducibility In Flow Cytometry

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Written By: Tim Bushnell, Ph.D.

Isaac Newton was famous for saying “If I have seen further than others, it is by standing upon the shoulders of giants.” Implicit in that statement is that the information that the giants provided was reproducible. In fact, reproducibility is central to the scientific method and as far back as the 10th century, the concept of reproducibility of data was being discussed by Ibn al-Haytham.

In 2011, Prinz et al. published an article that indicated a case study looking at reproducibility by Bayer Healthcare found only 25% of academic studies were reproducible. This was followed up in 2012 by a report from Begley and Ellis that indicated on 11% of 53 landmark oncology studies were able to be replicated. So it seems that while we are trying to see farther, our lens may be out of focus.

Bruce Booth, writing for Forbes, published an article called “Scientific Reproducibility: Begley’s Six Rules” and in this article, he proposed the following 6 rules that should serve as a roadmap in evaluating scientific work, both published and your own work. These ...

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From Purity To Biosafety, Understanding The Cell Sorting Process

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The Power Of Flow Cytometry In Cell Sorting

Often, cell sorting is described as trying to find a needle in a haystack. In 2008, David Brown wrote an article on cell sorting for the Washington Post. In that article, he wrote :

“Finding and sorting a few cells from a great mass of them is sometimes likened to finding a needle in a haystack. But it’s actually more watching a parade of 10,000 undertakers, and spotting the two who are wearing yellow ties, and the three who are wearing pink ties, and getting them out of the line, without making everyone else lose step.”

This really captures the essence of the sorting process. It is also a very scary process for the average researcher. After spending hours and hours preparing samples, they are often handed off to an operator who puts them into the cell sorter. With fingers crossed, the researcher hopes the cells are sorted correctly, and there are enough of them to perform the downstream experiments that are the real goal of the process.

Most of the time, things go right, but now and then, they fail, sometimes catastrophically. C ...

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