Flow cytometers and cell sorters were designed with blood cells in mind. This means that commercial cell sorters are optimized for sorting cells typically smaller than about 20 µm in diameter. However, it turns out that many cell types, including those of mammals, are larger than 20 µm. So what are your options if you need to sort large cells?
Flow Cell Tip Compatibility
The first issue is the size of the flow cell tip: Howard Shapiro’s
First Law of Flow Cytometry (found in his book
Practical Flow Cytometry) whimsically informs us that “A 51 µm Particle Clogs a 50 µm Orifice”. Therefore, you need to first make sure the flow sorter you are using has a flow tip larger than the diameter of your cells! As a practical matter, tip diameters are recommended that are 4-5 fold larger that the cell being sorted. Most commercial instruments now come equipped with 100 µm flow tips; some have catalog entries for larger tips (up to 200 µm in diameter). As a general rule, all manufacturers should be able to provide these much larger tips, although this may require a special order.
Droplet Formation
The second issue relates to the process of droplet formation during sorting. As mentioned in a
previous article, droplet formation in flow sorting is driven by an oscillator attached to the flow tip. The general theory of droplet production was worked out over 150 years ago by
Lord Rayleigh in England. He showed that droplets will only be produced if the frequency of the oscillator is lower than a specific value; this value is determined by the diameter of the flow tip and by the speed of the fluid coming out of the tip.
Here’s the thing; if we choose a large flow tip, the regular pressure of operation of the instrument (the sheath pressure) is too large, since it allows a very large amount of liquid to flow out of the tip, rapidly emptying the sheath tank. If we lower the pressure, this reduces the speed of the fluid coming out of the tip, but at the same time it reduces the highest frequency at which we can run the oscillator. This means our sort rate has to slow down.
Standard conditions have been worked out for the various flow tips and recommendations can be obtained from the instrument manufacturers. Reduction of the standard sheath pressures, using as low as 5-7 p.s.i., is recommended for the 200 µm flow tips. This reduces the maximum frequency of the oscillator to below 10,000 Hz, which is well into the auditory region; ear protection is highly recommended! The sheath pressure and oscillator frequency and drive amplitude are then adjusted empirically to get the most stable droplet breakoff possible. Different sorters have different “sweet spots,” so ad hoc experimentation is needed to find these.
Optimization is Critical
The third issue involves sort optimization. This is usually done with standard, indestructible fluorescent microspheres, asking the machine to sort a defined number of these beads then counting the numbers actually sorted. The machine settings (the droplet delay and phase adjustments) are then tweaked to get 100% recovery. It is important to use microspheres that are similar in size to the cells being sorted. Unfortunately, most commercial bead manufacturers only stock small microspheres (<10 µm). Happily, nature provides us with cheap, large and indestructible cells in the form of pollen and spores, up to 100 µm in diameter. These can be collected in the wild, or purchased from biological supply companies. Some are naturally autofluorescent and others can be stained using a number of different fluorescent dyes.
Finally, before sorting large cells, it is important to plan ahead. In the best back-of-the-envelope tradition, assuming we are using a 200 µm flow tip, with oscillation at 7,000 Hz, this means we can produce at best 7,000 droplets per second. To avoid “coincidence” effects, we generally arrange that there is around 1 cell per every 10 droplets. Our maximal possible sort rate is therefore only about 700/second and, should our desired cells represent 1% of the total population, then we will be only sorting 7 positives per second. Sorting 10
6 positives would take about 40 hours, which might well be impractical. Pre-enrichment strategies can get around this problem, which could also be avoided by designing experiments that require fewer sorted cells as the end-product.
Sorting More than Just Single Cells
What about much larger cells? Jet-in-air sorters have not been used routinely with flow tips larger than 200 µm. Sorting of larger objects requires different methods for sort deflection, including using air pressure as found in the
Union Biometrica COPAS series of sorters. In the
XL configuration, this instrument can sort objects as large as 1500 µm, with a trade-off in terms of sort rate. This allows sorting not simply of large cells, but of large clusters of cells including pancreatic islets, Drosophila embryos, plant seeds and early seedlings, and nematode worms.
One way or another, you are unlikely to feel out-of-sorts any time soon!
Notes on some instrument specifications:
BC MoFlo Astrios: 70 and 100 µm flow tips. Sheath pressure is adjustable from 4-100 p.s.i.
FACS Aria: 70, 85, 100, and 130 µm tips. Sheath pressure is adjustable from 5-75 p.s.i.
FACS Influx: 70, 86, 100, and 140 µm tips. Optional: 200 µm tip. Sheath pressure is adjustable from 1-90 p.s.i.
FACS Jazz: 100 µm tip only. Sheath pressure fixed at 27 p.s.i.
Originally published on March 4, 2013. Revised and updated on May 4, 2016.
David received his BA, MA and PhD degrees in Biochemistry from Cambridge University, and was a NATO Postdoctoral Fellow at Stanford University. He is Professor of Plant Sciences and Professor of Biomedical Engineering at the University of Arizona, and his research interests include biological instrumentation, developmental and tissue-specific gene expression in eukaryotes, functional genomics and proteomics, and issues in biodiversity.
