How To Analyze FACS Data And Prepare Flow Cytometry Figures For Scientific Papers
Written by Tim Bushnell, PhD
“It would be possible to describe everything scientifically, but it would make no sense; it would be without meaning, as if you described a Beethoven symphony as a variation of wave pressure.”
― Albert Einstein
The goal of any scientific process, as you know, requires the communication of the data that supports or refutes the hypothesis under testing.
Before it is deemed worthy of publication, it must survive the process of peer review ― where the data is laid bare before a group of experts in the field who judge the material impartially (usually) and in secret ― then pass judgment on the suitability of the information for publication.
The presentation of your data must be clear.
As such, choosing the right flow figures to communicate your data is essential.
Good handwriting (formerly known as proper penmanship) and drawings might have been enough to convince peers in the distant past, but not today.
Today, the expectation is that you’ll choose the right flow figures from all that are available, selecting the ones that reflect your data ...Read More
4 Core Techniques For Improving Fluorescence Activated Cell Sorting Results
Written by Mike Kissner.
Cell sorters have become more sophisticated to rival the multicolor capabilities of analytical cytometers, offering up to seven lasers and a score or more of detectors.
Likewise, cell sorting experiments have also become more complicated in terms of the number of markers and fluorophores utilized to identify target populations, and more complicated in terms of understanding an increasing canon of flow cytometry and cell sorting definitions.
These days, sorts of up to 12 or more colors are not uncommon.
While multicolor sorts are very feasible and can yield excellent results, success is always a product of very careful planning and optimization.
Attempting a 12-color sort of precious samples without trial runs will almost always result in failure and require a return to the drawing board to perform the optimization steps that were initially foregone.
Additionally, all setup steps, especially compensation, must be performed on-the-spot rather than during offline data analysis, as is commonly done in analytical experiments.
Satisfactory results require that all ...Read More
4 Critical Components In Cellular Proliferation Measurement
Written by Tim Bushnell, PhD
Cell proliferation is a critical component in biological systems.
While normal cell proliferation keeps the body functioning, abnormal proliferation (such as in cancer) can be a target for therapy.
Measuring how cells proliferate in response to a stimulus is a time-honored assay in science. This can be as simple as a cell count between untreated and treated cells. More sophisticated assays can include the use of 3H-thymidine or colorimetric assays (MTT assays).
While these all can measure proliferation, they lack the finesse that flow cytometry can bring to the assay – which allows the phenotypic identification of which cells are actually dividing, as well as allowing for calculations of values such as the precursor frequency, the percentage of cells that have divided, a proliferation index, and more.
Proliferation calls for cells to make a trip around the cell cycle, and there are many ways to measure cell division.
The focus here is on the long-term measure of cell division—the ‘temporal’ dimension for measuring such biological processes ...Read More
4 Spectral Viewers You Should Be Using For Your Flow Cytometry Experiments
Written by Tim Bushnell, PhD
At the heart of flow cytometry is the ability to make meaningful measurements of fluorescently tagged cells.
These fluorochromes can be bound to antibodies, a fluorescent protein, a reporter fluorochrome, and the like. Free online spectral viewers are useful in a variety of ways, all of which help improve experimental design and troubleshooting.
These spectral viewers have a special place on every scientist’s browser toolbar. I refer to them regularly, at least weekly.
During the process of panel design, it is useful to have these open to check and compare different fluorochromes. In fact, with recent upgrades to FluoroFinder, there is an integrated spectral viewer based on the filter configuration.
There are a host of different spectral viewers available online. Each one has its strengths and provides specific information. This often necessitates having to use two or three of them to get the information you want. These are the spectral links I use (in alphabetical order):Read More
The Difference Between Purity, Single Cell, And Recovery Cell Sorting Techniques
Written by Michael Kissner
If you have experience sorting different kinds of cell types on a droplet sorter, you may have noticed that sorting efficiency often seems closely tied to the cell and sample type.
For instance, you may have experienced a better outcome, such as a higher efficiency and more cells recovered, after sorting lymphocytes versus sorting an adherent cell line. There are some fundamental concepts that underpin this phenomenon, and understanding them will help you perform better cell sorting experiments.
What Is Flow Cytometry Cell Sorting Efficiency?
Sorting efficiency, in fundamental terms, is a real-time measurement, generated by the instrument, of how successfully its sorting system is able to resolve cells that we want to sort (target events) from cells we do NOT want to sort (non-target events).
Note that we are talking about the sorting system’s ability to resolve events here (the droplets) and NOT the electronics system.
Efficiency is calculated with the following equation:
The results of this equation are highly dependent on two aspects of the sort: 1) th ...Read More