Flow Cytometry Procedure For Accurate Sorting Of 5-10 Micron Cells

In the most common applications, cell sorters are utilized to purify cells on the order of about 5-30 microns in diameter, often human or murine leukocytes, or cell lines derived from liquid or solid tissue types.

Considering this typical size range, cells between 5-10 microns in diameter are typically the simplest cells to sort. That being said, the general rules of thumb in forming sample preparation, instrument setup, and sample collection are critical to generate sorts of high purity, recovery, and viability — the hallmarks of sort quality.

To this end, there are 4 steps you must take to ensure high quality when sorting 5-10 micron cells by flow cytometry…

1. Protect cell sample quality.

The tried-and-true adage of flow cytometry — garbage in, garbage out — should hover above and firmly guide each step of the sample preparation process for any kind of cytometry experiment, but is especially critical for sorting.

Poor sample quality will most surely guarantee sacrifices in purity, recovery, and/or viability, often to the extent of significantly, affecting the downstream experiment, so follow these guidelines for the best outcomes.

The key is to minimize debris because the amount of “junk” in a sample can often severely limit the purity, efficiency, and even viability of a sort.

As a reminder, the efficiency parameter reported by the instrument indicates how many target cells (cells in the sort gate) were sorted compared to how many target cells were thrown away in order to maintain high purity of the output.

The efficiency is highly dependent on event rate: the higher the event rate, the higher the chance that a non-target cell will fall close enough to a target cell that it may be sorted along with the target cell. These types of events are aborted (not sorted) in purity sort modes.

Even though debris particles often do not fall into the sort or even scatter gates, they do register as events. Since these events are not classified as sort events, any debris particle in the same droplet or close to a target cell’s droplet will result in an abort or coincidence of that target cell.

Debris can be minimized in the following two simple ways:

1. Keep cells alive during preparation.

Dying cells will lyse and fracture into debris. Keep cells cold during preparation, assuming the cell type permits. Avoid rough handling, especially when preparing a suspension of adherent cells.

Avoid scraping and expose cells to proteolytic enzymes for dissociation for the minimal amount of time necessary to remove cells from the growth surface or to disaggregate solid tissue.

1. Use a gradient or kit to remove debris if necessary.

Preparing cell suspensions from solid tissue can often generate debris. Centrifugation gradients or specialized debris removal kits can be a boon in removing unwanted particles from the sample.

Miltenyi Biotec offers a Dead Cell Removal Kit, for example, that works quite well. Disaggregated neural tissue can generate a lot of debris, and there are resources from Miltenyi and Worthington that are helpful.

2. Optimize flow cytometry instrument setup for cell type.

Instrument setup is not especially unique for cells in the 5-10 micron range compared to larger cells, so the usual guidelines for optimizing the instrument apply, including choosing the most appropriate nozzle.

Nozzles come in several different size ranges on cell sorters: 70, 85, 100, and 120-130 microns. The smaller the nozzle, the higher the pressure that is required to generate stable droplet breakoff the necessary distance from the nozzle orifice.

Larger or fragile cells may require lower pressures and larger nozzles to maintain purity, recovery, and viability of the sorted fraction, but many cell types in the 5-10 micrometer range can be sorted using the 70 micron nozzle and the higher pressure ranges required (~70 PSI).

The general rule that cytometrists follow when selecting a nozzle is: “the nozzle size should be no smaller than 5 times the diameter of the cell.”

Adhering to this rule, the 70 micron nozzle would be appropriate for cells up to 12-15 microns.

One nice benefit of the 70 micron nozzle is sort speed. The smaller the nozzle size, the faster that droplets can be generated.

Droplets effectively partition the stream into “sort units,” so the faster they are generated, the faster we can sort. The frequency of the droplet drive, which determines how many droplets are generated per second, can be as high as 90 kHz (90,000 droplets per second) at 70 PSI and can permit sample rates of ~30,000/second for sorting.

That being said, 70 PSI may be too high for some cell types in the 5-10 micron range. Try a larger nozzle and lower pressure if consistently poor viability is observed in the sorted fraction.

3. Always gate for single cells.

When setting up the sort, always include a doublet discrimination gate in the sort logic.

Doublets are events in which two particles pass through the laser spot so close to each other that they are classified by the instrument as a single particle and can contribute to low purity.

A doublet may consist of a target cell and a non-target cell, both of which will be sorted.
Because the instrument classifies both cells as one event, the presence of the non-target cell will not result in an abort.

A typical doublet discrimination gate plots the area pulse parameter against the height or width pulse parameter in a single channel, often forward scatter or side scatter (e.g. FSC-A x FSC-H). This allows the signal intensity to be discriminated from the time the particle spends in the laser.

Doublets often generate the same intensity in FSC and SSC as single particles but spend close to twice as long in the laser spot. It can be helpful to create two doublet discrimination plots, and gate hierarchically (SSC-A x SSC-H gated on FSC-A x FSH-H).

4. Keep cells alive and healthy during and after the sort.

Follow the simple steps below to ensure high purity, recovery, and viability of the sorted fraction.

1. Suspend the pre-sort sample in the right sample buffer for sorting.

Choose a suspension liquid that is most appropriate for your cell type, but avoid those containing CO₂-carbonate buffering systems, which are typical components of cell culture media.

These buffering systems are formulated for tissue culture CO₂ partial pressures and not atmospheric, so they can become alkaline over time and may contribute to cell death.

Media containing phenol red will turn purple under these conditions, indicating alkalinity.

PBS and HBSS, without calcium or magnesium, are good choices, and be sure to include some kind of protein, typically 1-2% FBS or BSA. Calcium and magnesium promote cell adhesion and stickiness, which is to be avoided at all costs. Protein helps keep cells viable and may prevent sticking as well.

1. Use a viability dye, always.

A viability dye like propidium iodide, DAPI, 7-AAD, or many of the other commercial or proprietary dyes, will not only help assess the cell health of the pre-sort sample but will also allow you to prevent sorting dead or dying cells along with viable cells.

Dead cells will shed markers or leak GFP, so they may appear non-fluorescent after the sort even though they appeared in the sort gate in the pre-sort sample, leading to an assessment of low purity. Keep in mind that the scatter plot is not always indicative of viability.

Cells that are dying may appear in the same population as the viable cells, while dead cells that have fractured will appear outside of the scatter gate configured for viable cells.

1. Never collect sorted cells in an empty tube.

Cells are traveling at many meters per second as they are sorted, and if they hit plastic at these velocities, they will be obliterated. Including a collection buffer in the collectIon tube will drastically improve post-sort viability.

The buffer can be the same buffer that is used to resuspend cells, but be aware that cells can be accompanied by significant volume, which can dilute the collection buffer.

If large numbers of cells are to be collected, one strategy is to use PBS or HBSS containing a high percentage of serum, considering that it will be diluted to normal concentrations once the collection tube is filled.

While sorting cells 5-10 microns in diameter does not present a particular challenge compared to other cell types, the standard procedures must be followed to ensure the hallmarks of a good sort — purity, recovery, and efficiency. These good practices, including protecting sample quality, optimizing instrument setup, gating on single cells, and always keeping your cells happy will guarantee quality sorts, time and time again.

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