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5 Steps To Improve Your Flow Cytometry Data Analysis

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Written By: Tim Bushnell, Ph.D.

With the continued emphasis on improving the reproducibility of scientific data, it is critical to remember that there is no single step that will solve this problem. Instead, it is a mindset that needs to be adopted. From adding in additional descriptors in the data to ensuring the proper information is extracted these steps, especially when communicated, can improve the robustness of your data. Further, as more and more automated analytical tools are being developed, using

Improving the quality of your data starts with how you approach your data analysis process, your experimental design and what happens when you sit down at the instrument.

1. Add keywords at the beginning of your experimental setup.

The flow cytometry standard file (FCS) is composed of two pieces – the listmode data, a spreadsheet of the values for each cell and a header filer – which is where the keywords are stored. These keywords are defined by the FCS standard and are automatically added by the instrument to the file. When troubleshooting an experiment, this is a go ...

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The Right Way To Read A Flow Cytometry Scientific Paper

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Written By: Tim Bushnell, Ph.D.

Scientific reproducibility and the public’s confidence in scientific results is critically important. One has to look no further than the website Retraction Watch to learn of the published papers that are being retracted for a host of reasons. Reasons for retractions can range from plagiarism to scientific misconduct. The cost of these retractions is more than losing a paper, it can lead to financial penalties and even prison. In March, 2019 Duke University settled a lawsuit brought by the US government for over 100 million dollars arising from fraudulent data being used by a researcher in grants and papers from that institution over the years.

These types of stories erode public confidence in scientific data and lead to issues like the discredited link between Autism and vaccination. If you couple this the issues of reproducibility that have been discussed in several articles like this one.

But right now science is facing a reproducibility crisis and the public’s trust in scientific results is faltering. As scientists, it is our responsibilit ...

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Avoid Data Loss By Following These Steps To Set Your Flow Cytometry Gates Correctly

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Written By: Tim Bushnell, Ph.D.

How are you defining your gates?

When you do primary analysis or single tube analysis, it’s important that you make sure your gates are set correctly.

You need to know that you can find the populations that you’re interested in so you can extract the appropriate data.

This is what allows you to do your secondary or statistical analysis confidently.

What Gates Should You Be Setting Up In Your Flow Experiments

At the beginning of the experimental design process, it is a good idea to sketch out a hypothetical gating strategy. This sketch will help evaluate the panel and ensure that those markers that need good resolution are visualized with brighter fluorochromes.

In any gating strategy, gates to address machine and processing issues should be used to exclude those events that could confound the analysis. These gates include a flow stability gate, a doublet discrimination gate, a ‘schmutz’ gate and finally a gate to eliminate dead cells and if a dump channel is in the panel, this is where it can be included.

Figure 1: Gating Strategy showing ...

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3 Ways To Measure Cell Death With Flow Cytometry

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Written By: Tim Bushnell, Ph.D.

Cell death is a natural part of the lifecycle of a cell. In cases of development, it is critical for the shaping of fingers during human development. The processes of ordered cell death, or Apoptosis, are so important that in 2002, Sidney Brenner, Robert Horvitz, and John Sulston received the Nobel Prize in Medicine for their work on understanding this process. There are many different ways to measure cell death and flow cytometry is an ideal tool for this technique. Whether you are just assessing the viability of your cells or you are interested in the exact stage of cell death your sample is in, there are a variety of ways that you can measure cell death.

The most basic reason to measure the end result of cell death is to ensure the quality of your cell sorting experiment. Sorting dead cells for downstream analysis is a waste of time and money. This will also yield spurious results.

Researchers developing new drugs that can kill cancer cells can use this technique to determine appropriate concentrations of drug to use, what combinations of drugs mig ...

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5 Essential Controls For Reproducible Fluorescent Microscopy Imaging

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Written By: Heather Brown Harding, Ph.D.

Fluorescent imaging has the potential to bring great insights into your research project.

But, there is a lot of room for error.

And one of the most common and completely preventable errors that many researchers make is not having the necessary controls.

Having all the necessary controls may seem tedious.

I know in graduate school I was not a huge fan of controls, and would sometimes put off doing all the controls I needed until the end.

However, doing your controls after the fact is a terrible idea.

You could realize that all the data you recorded was just an artifact and that you wasted your time and samples.

It’s incredibly important to plan out your controls and perform them ahead of time so that you know that you have specific staining and you’re not picking up random noise, auto-fluorescence, and non-specific staining, etc.

Here are 5 fluorescent microscopy controls that you should be including in all your experiments…

1. Unlabeled sample.

The first and easiest control to an unlabeled sample.

In an unlabeled sample ...

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