Polychromatic Flow Cytometry on the FACS Aria and LSR-II: The Emory Vaccine Center Strategy
The Emory Vaccine Center has recently acquired a FACS Aria and an LSR-II. This document is meant as a brief introduction to the Aria and LSR-II, and is particularly focused upon the development of polychromatic flow cytometry applications on these instruments. This document will evolve and grow, and I welcome any feedback by email. Note that parts of this discussion also apply to our MoFlo cell sorter.
Table of Contents
- Reagent availability
- Base configuration
- The Violet laser
- The Blue laser
- The Red laser
- Setting up the Aria and LSR-II Configuration
Reagent availability
With the introduction of new instruments capable of measuring >8 fluorescence parameters such as the FACS Aria and LSR-II cytometers from Becton Dickinson, and the Cyan from DakoCytomation, the instrument manufacturers have in some sense leaped ahead of the available reagents for these systems. Of course, the reagent manufacturers are working hard to fill the pipeline with reagents that can take advantage of these systems. Check out the links below, and contact your sales reps for more up-to-date information.
- BD
BioSciences: New Flurochromes for Multicolor Flow Cytometry. BD Biosciences
(Pharmingen and Immunocytometry Systems)have an extremely active program
to make new dyes available for their new instruments. In addition to their
off-the-shelf reagents, they have a custom conjugate
program (contact your sales rep). - Caltag: Check out individual antibodies for availability of new dyes
- Beckman/Coulter has a large number of antibodies labeled with PE-tandem dyes such as ECD (PE-Texas Red), PC5 (PE-Cy5), and PC7 (PE-Cy7) available.
- eBiosciences
It is also possible to prepare your own reagents to take advantage of these instruments. Mario Roederer's web site has all of the protocols, and is the bible in this area.
Base Configuration
The base configuration of the FACS Aria has 9 fluorescence detectors on three lasers, with the possibility of adding 4 more. The FACS Aria at the VRC is equipped with the full 13 fluorescence detectors, as shown in the diagram below.
The diagram, taken from the BD FACS Aria Users Guide, shows optical filters for the base configuration. Note the absence of optical filters on position C on each of the trigons, and in positions G and H on the octagon; PMTs in these slots—indicated in the lettered circles are also missing in the base configuration. In contrast, the FACS Aria at the Emory Vaccine Center is equipped with PMTs in all positions, though we have had to order filters from a third party (filters are available from both Omega and from Chroma).
The Violet Laser
There is little consensus on the choice of the best dyes to use on the violet laser. The choices can be seen in the following table:
| Relative Emission | Dyes | Comments |
|---|---|---|
| Shortest | Cascade Blue; Pacific Blue; Alexa 405 | The consensus seems to be evolving towards Pacific Blue, which also has the advantage of being significantly cheaper than the other two. Cascade Blue and Alexa 405 share the identical fluorophore; only the functional groups used to attach them to antibodies are different. |
| Middle | AmCyan; Alexa 430; Cascacde Yellow; Quantum dot | The consensus is evolving away from Alexa 430 towards AmCyan. In the future, it may evolve towards one of the quantum dots. |
| Longest | Quantum dot | Only quantum dots are excitable by the violet laser and emit at long wavelengths |
The normalized emission spectra of the Alexa dyes is shown in the figure below. Note that I am not displaying the excitation spectra, but these can be obtained on the BD Spectrum Viewer.
The filters included with the instrument were chosen for the features of these dyes. When using Alexa430 in the absence of a longer wavelength Qdot, it might be a good idea to use a much broader filter (e.g. a 550/100), taking care so that it doesn't overlap the blue laser line at 488 nm.
Alexa 405 and Pacific Blue are excellent dyes for use with the violet laser. They are reasonably bright, and not excited by the 488 nm laser, and require little compensation in other channels. In contrast, although Alexa 430 works, it has a number of drawbacks: (1) it is only suboptimally excited by the violet laser; (2) it is not very bright; (3) it is significantly excited by the 488 nm laser in addition to the violet laser; (4) it has a very broad emission spectrum and requires extensive compensation against FITC, PE, PE-Texas Red (or PE-Alexa610), and even against PE-Cy5 (or PE-Alexa647). I'm guessing that it would be the first label that I would drop if I didn't need all of the channels of the cytometer for an experiment. If it is used, it should be used on an antibody that binds with high affinity to a highly expressed cell surface protein, such as CD8.
Finally, we will be developing applications of Quantum Dots that we think may prove very useful in conjunction with the violet laser. If we are successful, the filter configuration on our violet laser will change significantly. Quantum dots can be synthesized to emit light across the visible spectrum.
The Blue Laser (488 nm)
The current configuration for the blue laser will accomodate labels as in the following table:
| Label | Detector | Dichroic Mirror | Filters |
|---|---|---|---|
| FITC; Alexa 488 | E | 502 LP | 530/30 |
| PE | D | 556 LP | 576/26 |
| PE-TexasRed; PE-Alexa594; PE-Alexa610 | C | 595 LP | 610/20 |
| PE-Cy5.5; PerCP-Cy5.5; PE-Alexa680; (PE-Cy5) | B | 655 LP | 695/40 |
| PE-Cy7; PE-Alexa750 | A | 735 LP | 760/60 |
We will be attempting to add a sixth color to this panel, as indicated in the following table:
| Label | Detector | Dichroic Mirror | Filters |
|---|---|---|---|
| FITC; Alexa 488 | F | 502 LP | 530/30 |
| PE | E | 556 LP | 576/26 |
| PE-TexasRed; PE-Alexa594; PE-Alexa610 | D | 595 LP | 610/20 |
| PE-Cy5; PE-Alexa647 | C | 635 LP | 665/40 |
| PE-Alexa700 | B | 690 LP | 720/45 |
| PE-Cy7; PE-Alexa750 | A | 735 LP | 760/60 |
Note that we are replacing the Cy5.5 tandems in the first table with the Cy5 tandem (or Alexa 647 tandem), in order to make more room between it and the PE-Cy7 (or PE-Alexa750) tandem. In between these two, we will attempt to use the PE-Alexa700 tandem. The emission spectra for the PE-Alexa dye tandems are shown in the figure below (note that for the Alexa700 and Alexa750, only the spectra for the small molecule dyes themselves were available).
In addition to buying what we can, we are preparing our own custom tandem dyes, using only a slight modification of the methods found on Mario Roederer's website, for preparation of the PE-Cy5 tandem. Our only modification is that we use "Reaction Buffer B" (100 mM carbonate, pH 8.4) during the modification of the phyobiliprotein with the NHS-derivatives of the Alexa dyes, instead "Reaction Buffer C" (500 mM carbonate, pH 9.0) that Mario uses for coupling of the cyanine dyes, which use different reactive group chemistry. I have not systematically evaluated the performance between the two buffers for the Alexa dye couplings, but we have verified that buffer B works, and we note that pH 8.3-8.4 buffers are often recommended for NHS couplings (for example, as in this PDF file from Molecular Probes).
Three more notes about the tandems.
- Our decision to focus on the Alexa dyes, rather than the cyanine dyes (Cy5, Cy5.5, and Cy7) was made relatively randomly. I don't yet know if it was a good choice or not.
- We have observed poor coupling of Alexa610 to PE, and this difficulty has been confirmed by Molecular Probes, who have suggested that we try Alexa 594 instead, which has a similar emission spectrum.
- Finally, antibody conjugates to all of the tandem dyes can be titered on the FACS Calibur using the FL3 channel, which employs a 670 LP filter (even the PE-TexasRed dyes, similar to the PE-Alexa610, can be measured in FL3, though for the life of me I don't really understand why this works). Data from the Calibur can even be used to determine if there is enough of the Alexa dye attached to the PE to obtain as much FRET from the PE to the Alexa dye as possible. At this point, I don't really know what will happen if you prepare tandems that are overcoupled to the Alexa dyes.
In addition to buying what we can, we are preparing our own custom tandem dyes, using only a slight modification of the methods found on Mario Roederer's website, for preparation of the PE-Cy5 tandem. Our only modification is that we use "Reaction Buffer B" (100 mM carbonate, pH 8.4) during the modification of the phyobiliprotein with the NHS-derivatives of the Alexa dyes, instead "Reaction Buffer C" (500 mM carbonate, pH 9.0) that Mario uses for coupling of the cyanine dyes, which use different reactive group chemistry. I have not systematically evaluated the performance between the two buffers for the Alexa dye couplings, but we have verified that buffer B works, and we note that pH 8.3-8.4 buffers are often recommended for NHS couplings (for example, as in this PDF file from Molecular Probes).
Three more notes about the tandems.
- Our decision to focus on the Alexa dyes, rather than the cyanine dyes (Cy5, Cy5.5, and Cy7) was made relatively randomly. I don't yet know if it was a good choice or not.
- We have observed poor coupling of Alexa610 to PE, and this difficulty has been confirmed by Molecular Probes, who have suggested that we try Alexa 594 instead, which has a similar emission spectrum.
- Finally, antibody conjugates to all of the tandem dyes can be titered on the FACS Calibur using the FL3 channel, which employs a 670 LP filter (even the PE-TexasRed dyes, similar to the PE-Alexa610, can be measured in FL3, though for the life of me I don't really understand why this works). Data from the Calibur can even be used to determine if there is enough of the Alexa dye attached to the PE to obtain as much FRET from the PE to the Alexa dye as possible. At this point, I don't really know what will happen if you prepare tandems that are overcoupled to the Alexa dyes.
The Red Laser
The current configuration for the red laser will accomodate labels as in the following table:
| Label | Detector |
Dichroic Mirror |
Filters |
| APC | C |
660/20 |
|
| Alexa680 (Alexa 700 is also possible) | B |
685 LP |
710/20 |
| APC-Cy7; APC-Alexa750 | A |
735 LP |
780/60 |
The original publications from Roederer and colleagues (1, 2, 3) on polychromatic flow cytometry used the following three labels run off of a red laser: APC, APC-Cy5.5, and APC-Cy7. We and others have found that it is possible to replace the APC-Cy5.5 tandem with a direct antibody conjugate of Alexa680. Although the excitation spectrum of Alexa680 suggests that it would not be optimally excited by the 633 nm laser, we have found that it provides a reagent with excellent brightness, and therefore we do not believe that it is worth the effort to produce an APC tandem. We've also heard that it is possible to use direct conjugates to Alexa 700, and that the emission spectrum of these reagents might better "fit" between the APC and the APC-Cy7 (or APC-Alexa750) tandem.
Setting up the Aria PMT Configuration
On an instrument like the FACS Calibur which does not have interchangeable filters, the meaning of the parameter names FL1, FL2, FL3, and FL4 are always the same. On instruments with changeable filters such as the FACS Aria, FACS Vantage, and the MoFlo, such names are often meaningless, as are the detector names like A, B, C.... Continued use of these names will violate the principle that FCS files should contain within them all of the information necessary to interpret the data without cross-reference to a notebook.
The FACS DiVA software that runs the Aria has an Instrument Configuration panel that looks like this (sorry for the low resolution):
