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How to Optimize Flow Cytometry Hardware For Rare Event Analysis

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“Not everything that can be counted counts and not everything that counts can be counted.” — William Bruce Cameron (but often misattributed to Albert Einstein)

What does this quote mean in terms of flow cytometry? Flow cytometry can yield multi-parametric data on millions of cells, which makes it an excellent tool for the detection of rare biological events — cells with a frequency of less than 1 in 1,000.

With the development and commercialization of tools such as the Symphony, the ZE5, and others which can measure 20 or more fluorescent parameters at the same time, researchers now have the ability to characterize miniscule population subsets that continue to inspire more and more complex questions.

When planning experiments to detect — and potentially sort — rare events using flow cytometry, we need to optimize our hardware to ensure that optimal signals are being generated and that rare events of interest are not lost in the system noise. This noise is also exacerbated by poor practices when running the flow cytometer.

There are 3 areas of hardware limitations that we ...

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3 Requirements For Accurate Flow Cytometry Compensation

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For those new to flow cytometry, compensation is confusing at best and terrifying at worst. Likewise, those who have been doing flow cytometry since the analog ages may be holding on to practices that, while suited to the analog instruments, should be left to the annals of history. As such, a lot of time is spent discussing compensation and the best practices for this critical process.

There are 3 rules that guide proper compensation, and they’ve been written about extensively since they first appeared in the “Daily Dongle” in 2011. It is always good to review and, importantly, there are some caveats and assumptions baked into the rules which bear closer examination.

Compensation Rule 1: “Controls must be at least as bright or brighter than the sample to which the compensation will be applied.”

To ensure that the correct compensation value is calculated, we need accurate measures of our controls.

We use the slope of the line between 2 populations with different intensities in the channel of interest to calculate compensation. In theory, that calculation should yield the sa ...

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5 Considerations For Statistical Analysis Of Flow Cytometry Data

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Congratulations, your grant has been funded! Now comes the hard part — performing the work that you are being funded to do. This means generating data and publishing papers. What was that hypothesis again? It must be in the grant somewhere, right?

For the sake of this blog, the grant is to study the effects of Cordilla Virus, which is known to cause lipid membrane flipping on CD8+ T-cells. This flipping results in phosphatidylserine expression on the outer membrane, causing infected cells to be phagocytosed by macrophages. A lead compound, Masiform D, has been identified that shows promise in reducing the viral load of patients infected with Cordilla Virus.

To avoid even the appearance of HARKing — Hypothesizing After The Results Are Known — it is important to start at the beginning of the statistical analysis process even before the first experiments are performed. This process consists of 5 steps:

1. Set the Null Hypothesis

The null hypothesis (H0) is a statement of what we think the state of the system is. In this case, the state of the system after treatment would be ...

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How To Choose The Correct Antibody For Accurate Flow Cytometry Results

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Next to the flow cytometer itself, the most important component of a flow cytometry experiment comes down to the antibodies. It is by using antibodies conjugated to fluorescent markers that we are able to identify our specific cells of interest and quantitate the amount of our target on the cell.

When I started in flow cytometry, I was immediately taken by the technology, and only later began to appreciate the importance of understanding what my reagents were and how they worked.

With the development and rise of monoclonal antibodies, each lab or group gave them a different name. This name could be the specific clone, where the antibody was harvested, or perhaps the target to which the antibody bound.

You might have attended a talk where one investigator discussed their studies on VLA-4, while a second might have discussed information obtained using Clone 9C10. These are both the same thing, but it was like the wild west out there.

This led to the development of the cluster of differentiation (or CD) nomenclature that we use today. First established in 1982 at the first Internationa ...

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How To Achieve Accurate Flow Cytometry Calcium Flux Measurements

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Most flow cytometry experiments work with antibodies conjugated to a fluorochrome for some variation on immunophenotyping. However, any fluorochrome that is excited by one of the available excitation sources, and emits within the range of the detectors, can be incorporated into an experiment.

One of the great pleasures of the past was leafing through the Molecular Probes handbook, seeing what fluorescent dyes had just been released, and thinking of possible applications for them. The classic example of non-antibody directed fluorochromes are DNA-binding dyes like PI, 7-AAD, and Hoechst, but there are many others.

Dyes exist for the detection of everything from large nucleic acids to reactive oxygen species, and from lipid aggregates to small ions. Concentrations of physiologically important ions such as sodium, potassium, and calcium can be important indicators of health and disease.

Calcium ions play an especially critical role in cellular signaling. As a signaling messenger, calcium is involved in everything from muscle contractions, to cell motility, to enzyme activity.

Cells tig ...

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