Zen and the Art of Fluorescence-activated Cell Sorting (FACS)

It’s Time to Evolve Your Consciousness

The day has finally come: you’re tired of sitting at the microscope with your Hunchback of Notre Dame lookin’ posture, haemocytometer, cell counter, and trypan blue in hand, clicking away. You look down at your ice box, unable to pull your eyes away from the source of the subconscious resentment that’s tearing apart your experiments. You keep counting, noting the mediocre cell viability and dreading the non-specific binding that is certain to ruin your day. A small brown bottle glints in the corner of your eye, its dark pigmentation conceals its vile contents but your most primal instincts, your reptilian hind-brain, is alerted and aware of what lurks inside. It’s none other than the plebeian choice of cell isolation reagent: magnetic beads.

A tear rolls down your face as you unpack the cell isolation magnet from the bottom cabinet. “I need an over-engineered solution that costs nearly a quarter of a million dollars and requires a full time skilled operator. I need something I can believe in.” You think to yourself, “…I need a flow sorter.”

What turn of fate has drafted you into being a lowly surf who uses antibody-coated magnetic beads to isolate your cell populations of interest? How can you escape the tyranny of your lower consciousness and transcend into realms of existential bliss and detachment from magnetic bead columns?

Many old buddhas, from Gautama to Bodhidharma, described such a state, and over the centuries countless eastern wisdom traditions, Zen and Mahayana Buddhism as well as the Gnostic mystics of the west, have struggled toward the experience of satori: freedom from magnetic bead purification. It was not until the late 20th century, however, that Zen reached its zenith in the form of the BD FACSAria II fluorescence-activated cell sorter.

Sorting Your Experiments Out of Saṃsāra

Saṃsāra is what the Zen Buddhist describes as the endless cycle of birth, meaningless existence, death, and rebirth, which is exactly how I would describe my various dissertation projects. Invariably, in any complex cell culture experiment I find myself suffering from impure cell populations and tedious purification procedures, and so last year I postulated that freedom from saṃsāra could only be attained via intense training in fluorescence-activated cell sorting (FACS). What follows is a highly technical decent into the workings of a typical cell sorter and an exploration of the existential questions raised by such a device. Proceed at your own risk.

A PhD student asked his adviser in all earnestness, “Does a cell have Buddha nature?”

FACS is similar to conventional flow cytometry in that cell populations of interest are first tagged with fluorochrome-conjugated antibodies specific to unique surface antigens. Internal antigens can also be tagged provided they are made accessible through a permeabilization procedure or genetically fused to fluorescent proteins such as GFP or mCherry. After washing away excess staining reagents the cells are resuspended in cold PBS or MACS buffer (PBS with some serum and EDTA mixed in for flavor), filtered to remove aggregated cells, and passed single-file (in theory) through a series of lasers that excite any fluorochromes present on the cell.

Figure 1. Simplistic FACS fluorochrome panel as depicted by the Biolegend Spectra Analyzer. Dark purple dotted/solid: Brilliant Violet 421 excitation/emission. Orange dotted/solid: PE excitation/emission. Pink dotted/solid: APC (allophycocyanin) excitation/emission. Purple, blue, green, and red vertical lines correspond to 405 violet, 488 blue, 561 yellow-green, and 633 red lasers, respectively.

Cytometers can have anywhere from 2 to 7 (or more!) lasers, flow sorters typically having more rather than less due to their already mammoth cost, that excite each fluorochrome optimally and stimulate the emission of secondary photons. The emission spectra for a given fluorochrome will always be red-shifted relative to the excitation frequency because between the moment when the exciting photon is absorbed and the secondary photon is emitted, the fluorescent molecule will lose some of its energy in the form of movement/heat. The distance between a molecule’s excitation and emission max frequencies is referred to as the Stoke’s shift [1].

Image result for Stokes shift
Figure 2. (A) Diagram of fluorochome energetic states. (B) Excitation vs emission spectra. Source: Bio-Rad

Emitted photons are initially diffracted through a prism, passed through several filters (the complexity of which an entire article would be necessary to describe), and finally collected by photomultiplier tubes (PMTs). PMTs are essentially funnels that convert photons to electrons via scintillation (sounds titillating, no?), and allow for gain to be applied through variable voltage control of the dynodes. Each laser typically has 2 to 4 PMTs associated with it.

PMTs have several downsides, including their physical size, and it should be noted that in some new cytometers PMTs are completely omitted and replaced with smaller semiconductor detectors. In the Cytek Aurora, for instance, each laser has upwards of 8 detectors thanks to their relatively tiny size.

Related image
Figure 3. PMT diagram. The photon (red) is converted to an electron through the process of scintillation, typically by a phosphor. The electron is then amplified via its interaction with each of the dynodes, with the voltage applied to the dynodes being proportional to the gain in the electronic signal generated by the PMT.

Besides the fluorescent signals of the surface antibodies, each cell is also characterized based on its forward and side scatter pattern by 2 dedicated PMTs associated with the blue (or sometimes the violet) laser. The forward and side scatter PMTs quantify the size and granularity of the cell, respectively (figure 4).

Image result for side and forward scatter flow cytometry
Figure 4. Forward and side scatter are associated with size and granularity of each cell, respectively. Source: Drexel University.

Finally, all of the scatter and fluorescent data is put together as a matrix of data that represents 1 cell. Elegant, no?

The adviser replied, “μ!”

Now all of this is just the workings of your typical, everyday flow cytometer. To actually sort the cells several more tricks must be employed.

We must first realize that, as enlightened flow sorting masters, we are sorting not the cells themselves but rather the charged droplets of liquid that they are encased within. Cells are segregated into individual droplets by the action of the nozzle, a tiny ceramic aperture with a diameter of typically 70 μm, 85 μm, 100 μm, or 130 μm.

The nozzle lies directly below the interrogation point, where the tagged cells and lasers mix and mingle, and through the black magic of electronics by the time the cell is segregated the fluorescent characteristics of the cell are recorded and a decision has been made by the software to sort the cell or not.

Below the vibrating nozzle lies the sort block which contains the deflection plates. Each charged droplet will pass through the sort block and if a droplet is thought to contain a particle of interest the deflection plates will become momentarily charged and deflect the droplet to any 1 of 4 positions. There are typically up to 4 deflection angles, 2 on the left and 2 on the right where a collection tube(s) can be placed, and any uncharged or uninteresting droplets fall straight into the trash can in the middle of the sort block.

Side note: if you set the laser delay wrong then every droplet, not just the droplets without your cells of interest, will fall into the trash. While this is a truly enlightening way to end a 6 hour sort, I would not recommend it to those who have not attended at least a 7-day anger management retreat at an ashram.

Theoretically, collection is the end of the sorting process and the each sort event is quantified by the software. Droplets of interest can be defined in any number of ways, and so the capabilities of a sorter are nearly endless.

Furthermore, sorts can be optimized for either yield or purity, and collection vessels range from 1.5 mL Eppendorf tubes to 96-well plates.

Meditations on Cell Sorting (So you don’t have to!)

  • Larger nozzle size = less pressure applied to your cells but slower sorting
  • Pre-filter your resuspended cells through a 0.2 um mesh immediately prior to sorting to prevent clogging of the nozzle. This is particularly important with 70 and 85 um nozzles.
  • Keep samples on ice and protected from light before sorting to prevent photo-bleaching of your fluorochromes
  • Use a fixable live/dead stain to exclude dead cells from your analysis. They stick to viable cells and bind non-specifically to antibodies and can form convincing, but unreal, populations of cells during analysis
  • Use compensation beads over live cells for compensation controls, they are more consistent. Be aware that there are antibodies that don’t bind well to them, though.
  • Beware of dyes that conjugate multiple fluorochomes such as PE/Cy5 and PerCP/Cy5.5. While useful, if not cared for correctly they will begin to dissociate and give signals in both component channels- messy!
  • Be aware of the brightness of each fluorochrome you use. Put the brightest fluorochromes on the dimmest (least expressed) antigens.
Source: Biolegend
  • Pre-add around 200 uL of serum-containing PBS to your collection tubes and vortex with the cap on. The static charge on the plastic of the tube will repel your tiny charged cell droplets and cause them to bounce out of the tube. Serum proteins will coat the plastic and neutralize the charge. Also, the volume gives them a nice little pool to land in.
  • A Zen poem for the road: “The old Eppendorf tube, a cell sorted in; plop!”

Final Thoughts

A helper T cell and a memory B cell are traveling through the lymphatic system when they come upon a soluble viral coat protein coated with iC3b. The B cell pinocytoses the viral protein and presents it via MHC II. The T cell, knowing that B cells should not pinocytose antigen unless it binds specifically to the B cell receptor thinks about it disapprovingly as they continue along. Eventually, they reach the draining lymph node. The B cell hands off the viral protein to a subcapsular macrophage and the T and B cell again continue on their journey. The T cell continues to ruminate on the odd behavior of his companion. A few hours later as they approach the thoracic duct the T cell can no longer contain himself and says to the B cell, “You know as a memory B cell you are not supposed to pick up non-specific viral proteins.” The B cell replies, “I put down the viral protein hours ago. Why are you still carrying it?”

References

  1. Banwell C.N. and McCash E.M. Fundamentals of Molecular Spectroscopy (4th ed., McGraw-Hill 1994) p.101 and p.113 ISBN0-07-707976-0
  2. BD FACSAria IIu User’s Manual. BD Biosciences.

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