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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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How To Use A Threshold To Reduce Background Noise In Flow Cytometry

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Written by Tim Bushnell, Ph.D

On most flow cytometers, the photomultiplier tube (PMT) is the interface between the fluidics system and the electronics system. It is the PMT that converts the photons emitted from the fluorochromes into the electronic current that is digitized and ultimately converted to the value stored in the listmode file.

Any stray photon of light or random electron emission from a dynode will cause a cascade, and ultimately a photocurrent. This is often known as dark current. The figure below shows the idealized idea behind this concept.

Dark current signal noise in a flow cytometer

Figure 1: Stylized signal coming off a PMT showing the dark current and actual signals of cells passing the laser intercept.

As this figure shows, if each of these peaks is counted, there would be over 50 ‘events’ seen by the flow cytometer. Most of these events would be considered junk or debris.

Imagine if each of these events was recorded in the listmode file — how large would the file be?

To reduce this background noise in the system, we can use something called a threshold.

The threshold is a value that the signal mu ...

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How To Set And Monitor Optimal Voltages For A Flow Cytometry Experiment

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Written by Tim Bushnell, Ph.D

When a researcher first sits down in front of the flow cytometer, they are faced with several choices.

One of the most confusing is, “What voltage do I set my detector at?”

Unless the researcher is running a fixed voltage system (Accuri, and others), this choice can dramatically impact the sensitivity of the instrument, making or breaking the experiment.

In the days of the analog flow cytometers, voltages were set by placing a quadrant gate on a bivariant plot, with the lower left quadrant encompassing the first log decade. Unstained cells would be run on the instrument and the PMT voltage set until these cells were contained within that lower left quadrant.

Flow Cytometry voltage settings using old school methods

Figure 1: Schematic of setting voltages ‘Old School’.

Because of how the data was processed in older generation instruments, a portion of the population was ‘off-scale’ and accumulated in the first channel.

Using this method, the highly autofluorescent cells would drive the voltage, often causing a compression of the less autofluorescent cells on the axis. However, this was not always ...

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Strengths And Weaknesses Of Isotype Controls In Flow Cytometry

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Using isotype controls in flow cytometry

Written by Tim Bushnell, Ph.D

Controls are critical for minimizing the effects of the variables in a scientific experiment so that the effect of the independent variable can be accurately measured.

In flow cytometry, there are a host of important controls necessary to properly interpret data generated in these experiments. Some of these controls include compensation, fluorescence minus one (FMO), stimulated and unstimulated, reference, and controls.

When it becomes time to publish, the proper use of these controls is critical in convincing the reviewer and reader that the data has been properly analyzed.

The isotype control is an experimental control where a sample is stained with an irrelevant antibody with the same isotype as the target antibody. Cells are gated and positivity is set based on the background staining of this isotype control.

The use of the isotype control to set negativity remains a topic of discussion and can confuse the novice to flow cytometry, especially when a reviewer may request why these controls were not included in a submitted paper.
Overall, the isotype Read More



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