What Is Fluorescent Activated Cell Sorting And 4 Other Questions About FACS Data Analysis

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 now known as the “Fluorescence Activated Cell Sorter”, or ‘FACS’ machine.

This first instrument had a single laser and two detectors, capable of measuring one fluorescence and ‘forward scatter’.

With advances in areas of electronics, lasers, optics, and fluorochromes, instruments are now available that can measure as many as 15+ simultaneous fluorochromes and sort at rates of 20,000 events per second.

Cell sorting technology has come a long way, but many scientists still struggle to answer basic questions about FACS analysis. Here are the 4 most common FACS-related questions…

1. What is FACS and how does it work?

The term FACS is held as trademark by BD Bioscience, but the word has become accepted as a reference for any cell sorter, regardless of vendor.

FACS combines the traditional power of flow cytometry and couples it with the ability to isolate the cells of interest.

The most common FACS systems on the market use electrostatic separation, although there are some systems that use a physical or microfluidics design for isolation of the cells.

Just about every cell sorter is also a standard flow cytometer. As such, cells are stained following standard methods and introduced into the sorting machine by gentle pressure.

From there, the cells undergo hydrodynamic focusing and flow, single file, towards the laser intercept point(s), as the below figure shows. 

fluorescence activated cell sorting facs | Expert Cytometry | facs data analysis

Next, the flow stream is vibrated at some frequency, breaking it into many thousands of droplets. Some of these droplets contain the cells of interest. It is to these droplets that an electric charge is applied.

As the droplet flies free, it enters an electrostatic field and based on the applied electric charge, is deflected to a collection tube. Those droplets that do not get a charge are discarded as waste.

There are some technical differences between the various electrostatic sorters on the market. These differences are predominantly based on where the cells are interrogated.

2. What are the range of cell types that can be sorted by FACS?

The cell type that can be sorted is limited to the size of the cell, the quality of the instrument, and the ingenuity of the investigator.

Cell sorters have a nozzle, and the size of the nozzle dictates how large (or small) a cell can be sorted. Most often, cells should be 4-5 times smaller than the nozzle being used.

Most sorters on the market today can sort from very small cells (bacteria) to very large cells. There is even a special sorter that can sort very large clumps of cells and even small organisms.

3. How fast can a FACS instrument process cells?

When it comes to the processing speed of a cell sorter, there are two points to consider.

The first point to consider is the inverse relationship between the size of the nozzle and the frequency of droplet generation that will produce a stable stream.

fluorescence activated cell sorting facs | Expert Cytometry | facs data analysis

The below table shows the frequency of sorting for several different nozzle sizes. You can see that there is a range of frequencies, which are related to the pressure of the system. The pressure of the system has to be balanced with the nozzle size to produce a stable stream.  

The second point to consider regarding the speed of the cell sorter is related to how many events per second the system should run. This relates the need for purity of the sorted product and the poison distribution of events within the fragmented stream.

fluorescence activated cell sorting facs | Expert Cytometry | facs data analysis

If there are too many events based on the frequency, this leads to the decreased purity and loss of recovered cells

fluorescence activated cell sorting facs | Expert Cytometry | facs data analysis

As the above figure shows, there is a greater chance of having two cells next to each other, or multiple cells in one drop, when the event rate approaches the frequency of droplet generation. A good rule of thumb is an event rate at ¼ the frequency, as the below table shows.

Now it becomes possible to calculate how long a sort might take. For example, sorting at 60 kHz, at a rate of 15,000 events/second, if one needs 100,000 cells for a downstream application, and the cells are at a frequency of 1%, will take at least ((100,000 cells)/(frequency))/15,000 about 667 seconds or 11 minutes for this sort.  Assuming a 50% recovery would double the number of input cells needed, thus increasing the time to 22 minutes or so.

4. What topics should someone new to cell sorting consider?

There are several important tips that can help a researcher who is new to cell sorting and help ensure the best possible outcome for the experiment…

  1. Talk to the operator(s) of the cell sorter. They are friendly and will be able to provide a wealth of information on planning and executing the experiment. Enter into their good graces by making them part of the process to ensure they care about your cells as much as you do.
  2. Review the protocol. Go over the staining protocol and make sure everything is ready before beginning the process. Do the back of the envelop calculation to make sure you know how many cells will be needed. Always assume a 50% loss from the cell sorter (due to electronic aborts, coincident events, cells dying post-sort, etc.).
  3. Coat the tubes. Coating your experimental tubes goes a long way to ensure that the charged droplets don’t stick to the plastic of the catch tube. Neutralizing that charge by coating with some protein can improve recover post sort.
  4. Filter the cells. Nothing ruins a sort like a clog. Remember Howard Shaprio’s First Law of Flow Cytometry – “A 51 𝞵m particle clogs a 50 𝞵m orifice.” Filtering the cells just before they are put on the sorter is a good way to minimize this issue. Another great trick is to add some DNAse (10 units per ml of sample) to help reduce clogging caused by dead cells releasing DNA (the biological equivalent of duct tape).
  5. Use the right controls. As with every flow cytometry experiment, controls are critical. Bringing a tube and saying ‘sort the green or red ones’ doesn’t endear one to the sort operator. As such, consider the following controls…
    1. Compensation controls
    2. Any gating specific controls (i.e. FMOs)
    3. Any controls necessary for setting gates
    4. (Paper control) – A copy of the gating strategy

6. Be on time. Sorting facilities often have back-to-back bookings, and need to get each one started on time. Be considerate to everyone and be on time.

In the end, cell sorting is a powerful tool that can be used to phenotypically identify cells of interest, from GFP+ transfectants to rare stem cells, and isolate them to homogeneity for downstream applications ranging from culturing, to genomics and NGS sequencing, to proteomics, etc. From the humble beginnings of a hybrid technology to the instruments available today, FACS analysis is now the entry point for many experiments. Understanding the inner workings of FACS instruments and the best practices for preparing samples will lead to more successful experiments.

To learn more about getting your flow cytometry data published and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on the Flow Cytometry Mastery Class wait list.

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Tim Bushnell, PhD
Tim Bushnell, PhD

Tim Bushnell holds a PhD in Biology from the Rensselaer Polytechnic Institute. He is a co-founder of—and didactic mind behind—ExCyte, the world’s leading flow cytometry training company, which organization boasts a veritable library of in-the-lab resources on sequencing, microscopy, and related topics in the life sciences.

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