By Michael Kissner
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.
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