What is it?
The FACSAria IIIu, or Aria as we more commonly call it, is a three-laser cell sorter.
What can it do?
The Aria has four lasers, 405, 488, 561 and 633 nm, and can detect light in sixteen photomultiplier tubes. Its primary function is to examine complex populations of cells and yield pure populations of cells. The Aria can sort lymphocytes at a rate of approximately 15,000 events per second. For larger cells, the rate is much slower in order to optimize the passage of cells through the instrument. Cells from single populations can be sorted into 96-well plates. Alternatively, the Aria can sort into two 15-ml tubes or four 5-ml tubes. The Aria can purify samples that are simply positive and negative for a single fluorophore or as complex as nine-color samples with intricate gating schemes.
Thanks to a renovated biosafety room within the FCCL facility, we are able to sort human samples. Details regarding the standard operating procedures for this can be found on the FCCL website. Briefly, certification that the samples are unlikely to contain risk groups 3 or 4 pathogens is required.
How does it work?
The Aria sorts by incorporating cells from the sample tube into a stream of sterile PBS. The stream is interrogated by lasers at the flow cell. The instrument electronics know the whereabouts of each cell as the cell passes through the laser intercepts and determines the specific cells meeting the sort criteria. Fig. 1 shows the image of the droplets generated by the Aria during a sort. A transducer in the instrument vibrates the stream inducing droplet formation. Cells in the stream are incorporated into the droplets. If a cell meeting the sorting criteria is in the last drop before the break off, the instrument will charge that drop and that charged droplet would be deflected into the proper collection tube by the charge plates.
We can adjust the intensity of the vibrations by changing the amplitude and the frequency of droplet formation by adjusting the frequency. The best sorting conditions are high pressure coupled with high frequency. Unfortunately, large cells require low pressures and suboptimal frequency in order to prevent damage to the cells. The Drop 1 and gap measure the location of the drops and ensure that the drops form properly. The instrument calculates a "sweet spot" and variation from this location would be indicative of a failure and cause the instrument to stop sorting.
To purify cells, it is critically important that cells remain in single cell suspensions, as clumps will easily clog the instrument. In addition, if multiple cells are incorporated into a droplet, the instrument will reject both cells, decreasing your yield from the procedure.
A common question that arises in planning a sort is how does the size of the cell affect the quality of the sort. It is important to understand that most flow cytometers are designed to sort lymphocytes, which are small uniform cells. The Aria is capable of obtaining quality results with other cell types, but conditions need to be carefully established. As a general rule of thumb, the size of the nozzle, the opening through which the stream will pass, should be five times larger than the cell size. For cancer stem cells, which can be approximately 15-20 mM in diameter, the 100 mM nozzle is typically sufficient, although some users prefer the 130 mM nozzle. When using large nozzles, it is difficult to obtain a steady stream of droplets. With large nozzles, the pressure under which the stream flows must be very low, in fact the ideal pressure for the 130 mM nozzle is lower than the Aria is capable of reliably sorting. For this reason, it takes longer to establish the stream and the cell yield is typically poorer than sorting with smaller nozzles. Reducing the nozzle to 100 mM allows us to increase the pressure and generate a more reliable stream.
Unfortunately, the only way to ascertain the best sorting conditions for your cells is to work out the conditions. Just as you optimize the number of cells and other conditions for your Western blot, RNA analysis, and other assays, we need to optimize the conditions for your sort. It obviously takes time and resources to optimize your conditions, but the quality of your experiments will benefit. If you sort the same cell type routinely, your future experiments will thank you for a little investment up front.
FAQs about the Aria?
Can the Aria detect stem cell side populations? Without a near-UV laser, we cannot identify cancer stem cell side populations using Hoechst 33342, but we can substitute the dye, DyeCycle Violet (DCV), and use the violet 405 nm laser. The distinct side population is defined in a multi-dot plot analysis of the Red (650 LP) vs. Blue (450/40 BP) emission of DCV, excited by the violet laser (405 nm). We have references for this technique, if you are interested.
What is the best purity expected? Best post-sort purity is obtained when the cells to be sorted are a discrete, bright population. Thus, the answer to this question can vary tremendously.
How is purity and yield balanced? We can bias the instrument to sort for greater recovery or better purity.
My cells have low expression of our protein. Can the Aria sort these cells? Marginally fluorescent populations are difficult to sort. Sorting works best when the population to be sorted is much brighter than its negative control. We advise setting very tight gates on dim cells. Dimly stained cells have fewer attached fluorochromes for the lasers to excite, leading to fewer photons gathered by the light collecting optics. Your yield is likely to be low.
Can the Aria sort cells expressing fluorescent proteins? The Aria is excellent for sorting GFP-transfected cell lines as GFP is maximally excited by the 488 nm laser. The past decade has seen the rapid development of fluorescent proteins. The red protein mCherry is a monomeric fluorescent protein with many advantages; it is optimized for expression in mammalian cells, the protein matures quickly after gene expression (half-life = 15 minutes), and it is photo-stable and resistant to photo-bleaching. Unfortunately, mCherry is best excited by a green to yellow laser line but can be excited by the blue 488 nm laser. DsRed is very similar to mCherry. Fig. 2 shows the pre-sort and post-sort analysis cells co-expressing GFP and DsRed.
Are there any other technologies that could serve a similar purpose?
The other instrument that can be used to purify cell population that is housed within the FCCL is the RoboSep. This fully automated instrument uses magnetic bead-based technology in which antibodies against cell markers are conjugated to magnetic beads. The instrument can then separate cells bound to beads from those that are not. Thus, populations can be separated using positive or negative selection. The RoboSep is particularly well-suited when purifying a single population of cells that can be identified with a single marker.