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5 FlowJo Hacks To Boost The Quality Of Your Flow Cytometry Analysis

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Written By: Tim Bushnell, Ph.D.

Primary data analysis, that is the analysis at the sample or tube level, is where the populations of interest are identified and the necessary data is extracted for secondary analysis. Since the creation of the FCS standard, flow cytometrists have had the ability to analyze data in third party software because of the communities agreement on the standard. The most recent standard of FCS3.1. The FCS standard divides the file into two components, the listmode file that contains the sequential data from all the detectors. The header file contains what are termed ‘keywords’. These include define keywords that are added to the file automatically, as well as terms that can be defined by the user.

These keywords are only the tip of the iceberg when it comes to performing advanced analytics. This article will focus on the power of FlowJoX (FJX) and provide some tips and tricks to improve the researchers analysis.

1. Embedding and using keywords.

The header information of the FCS file contains a great deal of information that is very useful to review and e ...

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5 Special Considerations for Live Cell Imaging

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Written By: Heather Brown-Harding, Ph.D.

Live cell imaging is advantageous for research were you may be worried about artifacts of fixation or when you want to measure a phenomenon over time. Live cell imaging is more difficult to achieve than fixed samples because we need to keep the cells live AND happy along with obtaining the images we need. We can reduce artifacts by keeping the cells in a favorable environment and minimizing external stressors. Here are 5 points to keep in mind when setting up your live cell imaging experiment.

1. Environmental controls.

A specific and controlled environment needs to be created for successful live cell imaging. Specifically, with mammalian cells, we need a specific temperature, CO2 concentration, and humidity. The temperature should be 37 degrees Celsius, CO2 should be 5%, and there needs to be enough humidity so that we don’t have evaporation. We are essentially trying to recreate the environment of the incubator for the cell, while we are imaging.

Unfavorable conditions can cause artifacts, or cell death, defeating the purpose of live ...

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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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