Increase Cell Viability With These 3 Flow Cytometry Experimental Research Design Tips

The cell sorting process, while generally well-tolerated by most cell types, is inherently stressful.

Cells are first manipulated in suspension for up to several hours to prepare and stain them.

Then, during the cell sorting process, these cells are pushed through narrow tubing under high pressure in the range of approximately 10-70 PSI, rapidly depressurized after passing through a nozzle, and then jetted through the air at velocities of 20 m/s (~44 MPH) or higher.

Keeping cells healthy, happy, vital, and viable over the course of a cell sorting experiment is important, not only to keep cells alive during the sort, but also so that the recovery of cells from the sort is high.

Cells that die before, during, or after the sort will likely not be counted during a recovery assessment, leading to an unacceptably low cell output.

Taking into account the considerations described below when designing a cell sorting experiment will add a layer of robustness to your protocol to ensure that cells are healthy over the entire course of a cell sorting experiment—from preparation, to sorting, to collection.

First, you must suspend your cells in the right buffer and keep them at the right temperature. However, this is just the beginning. Here are 3 more experimental research design tips you must consider if you want to keep your cells viable…

1. Choose the appropriate instrument settings for your cell types.

Cell sorters are generally equipped with multiple nozzle sizes that each requires a specific pressure range for proper operation. Both the nozzle size and the pressure are additional parameters that may make the difference between a viable or dying/dead cells after the sort.

The nozzle sizes available on a cell sorter are generally 70, 85, 100, 120 or 130 mm in diameter. Some sorters can be configured with even larger nozzles in the range of 150 or 200 mm. The bigger the cells you are sorting, the larger the nozzle you need. This is the case for a few key reasons.

Firstly, cells that are too large for the nozzle and droplet size may cause perturbations in droplet formation. This instability may then cause a phenomenon called fanning, which will result in unstable sort streams that appear to spray in a variety of angles rather than the angle required to deposit the cells in the center of the collection tube.

Fanning can significantly reduce viability and yield by the improper deposition of cells and possibly to increased shear forces as well.

Secondly, a larger nozzle may be advantageous due to reasons related to pressure. The larger the nozzle size, the lower the pressure under which the sorter will be running. Larger or delicate cells that are more fragile may require a lower sheath pressure to remain healthy. The rule of thumb in the field is to choose a nozzle size that is at least five times the diameter of the cells. For example, if you plan on sorting 20 mm cells, be sure to choose the 100 mm nozzle or bigger.

The differences in pressure can be significant between nozzle sizes, so keep this in mind as well when choosing a sort setup. While the 70 mm nozzle is commonly set up under 60-70 PSI conditions, this drops to between 20-30 PSI when the 100 mm nozzle is installed. In addition to the overall system pressure, the sample pressure, or sample flow rate can also be important to maintaining viability.

Delicate cells may be healthier if sorted under a lower flow rate to minimize the impact of shear forces.

Incidentally, samples should be run at lower flow rates on sorters on which the cells are interrogated in a channel (typically a cuvette). On these kinds of systems, the velocity of sheath is parabolic across the flow cell – in other words, the sheath flows slower towards the walls of the cuvette than it does in the center.

Keeping the sample pressure low ensures that the core stream, through which the cells flow, is narrow and that its velocity is consistent. If the differential pressure is high, cells traveling in the outer portion of the core stream will arrive at the droplet break-off with different timing (drop delay) than cells traveling in the center of the core stream, resulting in a lower-than-expected recovery.

Additionally, make sure droplet charging is set up properly on the instrument so that fanning is minimized. The parameters to do so vary based on the instrument, but every instrument will have steps during its setup to make sure that the side streams generated during the test pattern are tight and stable.

Finally, dirty samples with high proportions of debris or dead cells may also contribute to fanning side streams, so sample preparation and quality is a related parameter to keep in mind as well.

2. Capture your cells gently.

Since cells are traveling so fast during the sorting process, it is critical that they are captured in the collection tube as gently as possible.

Most importantly, collection tubes must have some kind of liquid in them, a “capture medium”, into which the cells are directed.

Otherwise, the cells will be deposited on the plastic of the collection tube at high velocity, and most cells types will typically be obliterated upon this kind of impact, severely compromising viability.

There are several options for a capture medium. Cell culture medium buffered with a CO2-carbonate system is NOT a good choice for pH-related reasons. A common choice is the same buffer that is used for suspension of cells during the sorting process. However, it is important to keep in mind that there may be significant dilution of this buffer, depending on the nozzle size and number of cells sorted.

Most of the volume of liquid that is deposited in the collection tube is actually sheath – the cell itself comprises a very small portion of each droplet. Moreover, the larger the nozzle size, the more sheath each droplet will contain, as a function of the cube of the radius of the droplet. Therefore, sorting under 100 mm nozzle conditions may dilute the capture medium roughly three times more than sorting under 70 mm nozzle conditions.

To this end, it is common to formulate the capture medium with this dilution in mind. Many researchers choose to sort into PBS + 50% serum with the expectation that the serum will reach a final concentration when the sort is complete.

Additionally, sheath fluid tends to layer on top of the collection medium during the sort. Over a long sort, the cells will thus effectively be suspended in sheath fluid (saline) for an extended period of time, which may have deleterious effects on viability. In such situations, it may be advantageous to pause the sort, remove the tube, and agitate to mix sheath and capture medium.

It is not sufficient to solely supply the cells with capture medium in the tube—it is also extremely critical to ensure that the cells are directed into the capture medium and not onto the plastic walls of tube, where they will be destroyed on impact or eventually dry out and die.

When setting up the sort, ensure that the operator has adjusted the side stream deflection so that the droplets are injected into the center of the tube at the initial level of the collection medium. Sometimes, this angle may not necessarily be equivalent to the angle that directs the side streams into the center of the mouth of the tube; observe also where the side stream interacts inside the tube and that this interaction will place them at the surface of the capture medium.

The deposition of droplets under “test pattern” or “test sort” conditions may be slightly different than that under actual sorting conditions. This has to do with the number of droplets that are deflected during each sort event. A test pattern or test sort typically sorts with a “one drop envelope”—in other words, each sort event will contain one and only one droplet.

However, under actual sorting conditions, a sort envelope may contain one or two droplets in order to increase yield by ensuring that those cells dispersing close to the droplet borders are captured. It turns out that a sort event with two droplets will be deflected slightly differently than a sort event containing one droplet due to differences in aerodynamic profiles. This can be observed on a sorter equipped with a camera above the collection tubes.

Sorting under “single cell” conditions, which always use a single drop envelope for counting accuracy, will produce tighter side streams than sorting under typical bulk “purity” sort conditions. While this effect will not have a significant impact on a typical sort, it is an interesting phenomenon that’s worth mentioning.

3. Always assess viability during the sorting process.

Do you know the viability of the cells before they are input into the instrument? If not, you should. The output of a sort is only as good as the input, so knowing the quality of the input is essential for assessing the output.

A nucleic acid-binding dye, like PI, 7-AAD, or many of the other dyes offered by reagent manufacturers are a better choice than the amine-reactive dyes for cell sorting because they provide an instantaneous real-time assessment of viability. Amine-reactive dyes, while an excellent choice for cell-analysis applications, are applied to the sample during staining and then washed out. Therefore, cells that die after the staining process will not be included in the viability assessment.

On the other hand, nucleic acid-binding dyes can be formulated as a component of the suspension buffer and will indicate dead cells essentially immediately. Cells that die during the preparation or during the sort will thus be included in the viability assessment.

Including a viability dye can also help explain poor purity after a sort. For example, cells that express unanchored, cytoplasmic GFP may release this protein when their membranes are ruptured as they die. These cells will thus appear GFP-negative after a sort, a viability dye and a viability-dye only will be able to distinguish these cells as dead and GFP- (that were most likely GFP+ when they were alive and passing through the interrogation point during the sort) rather than live GFP- cells that were sorted inappropriately.

Cells are remarkably hardy to be able to survive the cell sorting process at all. With minor adjustments for a particular cell type, the simple suggestions described above are tried and true methods to ensure high viability and robustness after sorting. Cell sorting has become commonplace for virtually all fields of biological research, so there is a precedence for sorting nearly every cell type. There are excellent cell sorting and flow cytometry resources for cell type or tissue-specific methods, but the tips above should provide solid groundwork for most cell sorting protocols.

To learn more about current best practices in flow cytometry, 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.

Join Expert Cytometry's Mastery Class
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.

Similar Articles

The Power Of Spectral Viewers And Their Use In Full Spectrum Flow Cytometry

The Power Of Spectral Viewers And Their Use In Full Spectrum Flow Cytometry

By: Tim Bushnell, PhD

What photon from yonder fluorochrome breaks?  It is … umm… hmmm. Let me see. Excitation off a 561 nm laser, with an emission maximum of 692 nm. I’m sure if Shakespeare was a flow cytometrist, he might have written that very scene. But the play is lost in time. However, since the protagonist had difficulty determining what fluorochrome was emitting photons, let’s consider how this could be figured out. In my opinion, one of the handiest flow cytometry tools is the spectral viewer. This tool helps visualize the excitation and emission profile of different fluorochromes, as well as allowing you…

3 Must-Have High-Dimensional Flow Cytometry Controls

3 Must-Have High-Dimensional Flow Cytometry Controls

By: Tim Bushnell, PhD

Developments such as the recent upgrade to the Cytobank analysis platform and the creation of new packages such as Immunocluster are reducing the computational expertise needed to work with high-dimensional flow cytometry datasets. Whether you are a researcher in academia, industry, or government, you may want to take advantage of the reduced barrier to entry to apply high-dimensional flow cytometry in your work. However, you’ll need the right experimental design to access the new transformative insights available through these approaches and avoid wasting the considerable time and money required for performing them. As with all experiments, a good design begins…

The Fluorochrome Less Excited: How To Build A Flow Cytometry Antibody Panel

The Fluorochrome Less Excited: How To Build A Flow Cytometry Antibody Panel

By: Tim Bushnell, PhD

Fluorochrome, antibodies and detectors are important. The journey of a thousand cells starts with a good fluorescent panel. The polychromatic panel is the combination of antibodies and fluorochromes. These will be used during the experiment to answer the biological question of interest. When you only need a few targets, the creation of the panel is relatively straightforward. It’s only when you start to get into more complex panels with multiple fluorochromes that overlap in excitation and emission gets more interesting.  FLUOROCHROMES Both full spectrum and traditional fluorescent flow cytometry rely on measuring the emission of the fluorochromes that are attached…

Flow Cytometry Year in Review: Key Changes To Know

Flow Cytometry Year in Review: Key Changes To Know

By: Meerambika Mishra

Here we are, at the end of an eventful year 2021. But with the promise of a new year 2022 to come. It has been a long year, filled with ups and downs. It is always good to reflect on the past year as we move to the future.  In Memoriam Sir Isaac Newton wrote “If I have seen further, it is by standing upon the shoulders of giants.” In the past year, we have lost some giants of our field including Zbigniew Darzynkiwicz, who contributed much in the areas of cell cycle analysis and apoptosis. Howard Shapiro, known for…

What Star Trek Taught Me About Flow Cytometry

What Star Trek Taught Me About Flow Cytometry

By: Tim Bushnell, PhD

It is no secret that I am a very big fan of the Star Trek franchise. There are many good episodes and lessons explored in the 813+ episodes, 12 movies (and counting). Don’t worry, this blog is not going to review all 813, or even 5 of them. Instead, some of the lessons I have taken away from the show that have applicability to science and flow cytometry.  “Darmok and Jalad at Tanagra.”  (ST:TNG season 5, episode 2) This is probably one of my favorite episodes, which involves Picard and an alien trying to establish a common ground and learn…

5 Flow Cytometry Strategies That Sun Tzu Taught Me

5 Flow Cytometry Strategies That Sun Tzu Taught Me

By: Tim Bushnell, PhD

Sun Tzu was a Chinese general and philosopher. His most famous writing is ‘The Art of War’, and has been studied by generals and CEOs, to glean ideas and strategies to help their missions. I was recently rereading this work and thought to myself if any of Sun Tzu’s lessons could apply to flow cytometry.  So I have identified 5 points that I think lend themselves to thinking about flow cytometry.  “Quickness is the essence of the war.” In flow cytometry, speed is of the essence. The longer the cells are out of their natural environment, the less happy they…

A Basic Guide To Flow Cytometry (3 Foundational Concepts)

A Basic Guide To Flow Cytometry (3 Foundational Concepts)

By: Meerambika Mishra

Mastering foundational concepts are imperative for successfully using any technique or system.  Robert Heinlein introduced the term ‘Grok’  in his novel Stranger in a Strange Land. Ever since then it has made its way into popular culture. To Grok something is to understand it intuitively, fully. As a cytometrist, there are several key concepts that you must grok to be successful in your career. These foundational concepts are the key tools that we use day in and day out to identify and characterize our cells of interest.  Cells Flow cytometry measures biological processes at the whole cell level. To do…

Which Fluorophores To Use For Your Microscopy Experiment

Which Fluorophores To Use For Your Microscopy Experiment

By: Heather Brown-Harding, PhD

Fluorophore selection is important. I have often been asked by my facility users which fluorophore is best suited for their experiments. The answer to this is mostly dependent on whether they are using a widefield microscope with set excitation/emission cubes or a laser based system that lets you select the laser and the emission window. Once you have narrowed down which fluorophores you can excite and collect the correct emission, you can further refine the specific fluorophore that is best for your experiment.  In this blog  we will discuss how to determine what can work with your microscope, and how…

4 No Cost Ways To Improve Your Microscopy Image Quality

4 No Cost Ways To Improve Your Microscopy Image Quality

By: Heather Brown-Harding, PhD

Image quality is critical for accurate and reproducible data. Many people get stuck on the magnification of the objective or on using a confocal instead of a widefield microscope. There are several other factors that affect the image quality such as the numerical aperture of the objective, the signal-to-noise ratio of the system, or the brightness of the sample.  Numerical aperture is the ability of an objective to collect light from a sample, but it contributes to two key formulas that will affect your image quality. The first is the theoretical resolution of the objective. It is expressed with the…

Top Technical Training eBooks

Get the Advanced Microscopy eBook

Get the Advanced Microscopy eBook

Heather Brown-Harding, PhD

Learn the best practices and advanced techniques across the diverse fields of microscopy, including instrumentation, experimental setup, image analysis, figure preparation, and more.

Get The Free Modern Flow Cytometry eBook

Get The Free Modern Flow Cytometry eBook

Tim Bushnell, PhD

Learn the best practices of flow cytometry experimentation, data analysis, figure preparation, antibody panel design, instrumentation and more.

Get The Free 4-10 Compensation eBook

Get The Free 4-10 Compensation eBook

Tim Bushnell, PhD

Advanced 4-10 Color Compensation, Learn strategies for designing advanced antibody compensation panels and how to use your compensation matrix to analyze your experimental data.