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3 Advantages FCS Express 6 Has Over Other Flow Cytometry Data Analysis Software Programs

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Written by Tim Bushnell, Ph.D.

After running a flow cytometry experiment and collecting the data, the first and only thing any scientist wants to do is analyze the data.

To do this properly in the field of flow cytometry, a quality data analysis software program is required.

The problem is, most of the current data analysis programs are archaic, clunky, or overly complex. The package or packages that were useful in the 1990s have not been updated properly and, as a result, are buggy. Worse, they often require a great deal of training to use correctly.

However, the biggest problem overall with the majority of the data analysis software programs available is their outdated workflow.

For example, most of these programs require you to follow this sequence: load your data into the software program, perform your gating and analysis in the software program, and then spend an immense amount of time exporting your data to PowerPoint, Excel, Prism, and so on, and then further analyzing your data.

…and further analyzing your data, and further analyzing your data, and further analyzing y ...

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4 Steps To Validate Flow Cytometry Antibodies And Improve Reproducibility

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Written by Tim Bushnell, Ph.D

Reproducibility is the name of the game in science.

For scientific results to be valuable, they must be reproducible. The current crisis in scientific reproducibility has been well-highlighted, and has spurred the NIH to initiate a reproducibility and rigor initiative for grant applications.

There is significant concern about the quality and consistency of antibodies in scientific research.

Bradbury & Plückthun published a letter in Nature, signed by over 100 researchers, that called for standardizing antibodies by moving away from reliance on traditional monoclonal and polyclonal production towards generation of recombinant antibodies.

They estimate that $350 million a year is wasted on bad antibodies in the US alone. They go on to estimate that to produce recombinant binding reagents to 20,000 human genes would cost about $1 billion — three years worth of wasted antibodies.

Until such robust reagents are available, it is the responsibility of the researcher to be vigilant in their use of antibodies.

This is especially critical in the realm of f ...

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Why Understanding Fluorochromes Is Important In Flow Cytometry

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Written by Tim Bushnell, Ph.D

At the most basic level, a flow cytometer is photon counting device. It captures the emitted photons from fluorochromes present on targets — be they cells, beads, or other particles.

These fluorochromes can be attached to antibodies or proteins (like Annexin), free molecules that become fluorescent when bound to a target (DNA dyes), or have different fluorescent characteristics under different biological conditions (Indo-1, JC-1).

Fluorescent molecules are the tools of the trade in flow cytometry and, with continued advances in chemistries, it is helpful to step back and review their essential properties.

Why Understanding The Jablonski Diagram Is Important

When a molecule absorbs a photon of light, an electron is promoted to a higher energy state. This excited state is not stable, and the molecule releases this energy in several different ways.

When the release of energy is an emission of light, it is termed fluorescence.

This process can be visualized by the Jablonski diagram, which is a useful way to model the electronic states of a compound. Such ...

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5 Steps For Accurate Flow Cytometry Statistical Analysis Results

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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 support them.

During initial experiment design, consider the following…

1. Power the flow cytometry experiment properly.

Simply put, the statistical power of an experiment is the likelihood that the experiment will detect an effect if there is one to be measured.

The higher the experiment is powered, the lower the chance of making a Type II (false negative) statistical error.

There are a variety of calculators out there and one of the most useful is Statmate, fr ...

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3 Guidelines For Setting Compensation Controls In Flow Cytometry Experiments

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Guidelines for setting flow cytometry compensation controls

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 colors measured within a single experiment, the more crowded the spectrum becomes and the more severe this kind of crosstalk is.

Compensation is a mathematical process that deals with this problem by removing a percentage of the total signal from each detector.

This percentage corresponds to the amount of signal spillover signal that is contributed to this detector by all the other fluorophores being used in the ...

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