What Is Fluorescent Activated Cell Sorting And 4 Other Questions About FACS Data Analysis
Written By Tim Bushnell, PhD
Prior to the mid-1960’s, the ability to study a defined cell type was severely limited.
Researchers had to use centrifugation methods, such as differential centrifugation, rate zonal centrifugation, or isopycnic centrifugation, to define cell types.
All of these methods would allow separation of cells based on the property of the particles within different separation medias, but didn’t allow for very fine resolution of the cell populations.
That all changed starting in the mid-1960’s, when Mack Fulwyler published the first true cell sorter, which combined the power of cell characterization by the Coulter principle with the electrostatic separation of droplets developed by Richard Sweet (and used in inkjet printers).
For the first time, researchers could rapidly isolate individual cells based on more precise physical characteristics.
4 Common Questions About FACS Analysis
Early cell sorting technology eventually found its way into the Herzenberg lab at Stanford University, where a talented research group added lasers and developed what is no ...Read More
4 Biggest Mistakes Scientists Make During Multicolor Flow Cytometry Cell Sorting Experiments
Written By Mike Kissner
Multicolor cell sorting is a complicated process and certain scientific errors can be common.
Unsuccessful multicolor sorts can result in erroneous data and inconclusive results. Successful multicolor sorts, on the other hand, can give excellent results and lead to dynamic conclusions.
Successful multicolor cell sorting requires special attention to planning.
Using specific setup strategies for your experiment can create a streamlined system for an otherwise complicated process. For example, these critical steps and strategies for multicolor sorting experiments can save you time and maximize your results.
When setting up a multicolor experiment, the most common mistakes are failing to set PMT voltages properly, failing to use a viability dye, failing to address doublet discrimination properly, and failing to set the right sort regions and gates. Eliminating these 4 mistakes is important for any kind of flow cytometry experiment, but particularly for flow cytometry cell sorting experiments.
The following 4 mistakes should be avoided prior to the setup phase, wh ...Read More
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