5 Gating Strategies To Get Your Flow Cytometry Data Published In Peer-Reviewed Scientific Journals
Written by Tim Bushnell, PhD
“Every block of stone has a statue inside it and it is the task of the sculptor to discover it.” — Michelangelo
When sitting down to perform a new analysis of flow cytometry data, it is much like Michelangelo staring at a piece of marble. There is a story inside the data, and it is the job of the researcher to unravel it.
The critical difference between sculptor and scientist is that while the sculptor is guided by a creative vision, the researcher is guided by very particular laws of nature and a specific method of working through a biological hypothesis to avoid shaping the results to his or her whims.
Science must be objective, or it is simply an exercise in creative sculpting, which does nothing to move science forward.
Thankfully, there are many ways to avoid shaping the results, and instead sifting for the real and actual data that is relevant to the flow cytometry experiment at hand.
Communicating the results of a flow cytometry experiment is where the researcher has the power to make new or subtle findings instantly comprehensible to the aud ...Read More
How To Create A Flow Cytometry Quality Assurance Protocol For Your Lab
By EditaMotyčáková, Ph.D.
Editor’s note: This is based on Edita’s experiences implementing the proposals reported in Perfetto et al., (2012) Nature Protocols 7:2067. She submitted this to the ExCyte Mastery Class and developed the Excel sheet (attached) to assist others in tracking their QC data.
Developing and implementing a new Quality Control (QC) protocol can sometimes be a daunting task.
With the continued emphasis on reproducibility in science, QC programs are an essential step that cytometrists are encouraged to both develop and implement.
This QC program comprises several assays that are focused on three important characteristics of the flow cytometer:
(1) Optimal instrument setting (e.g. instrument optimization)
(2) Cytometer sensitivity (e.g. instrument calibration)
(3) Monitoring of day-to-day variability in measurement (e.g. quality assurance)
QC Program Step #1 — Instrument Optimization
Instrument optimization assays include protocols useful for determination of laser power, photoelectron efficiency, testing of filter characteristics, evaluation of signa ...Read More
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