Cytometry and Antibody Technology

Spectral Flow Cytometry

Work in progress
Spectral Cytometry: The Basics

In conventional cytometry, each detector is assigned to a fluorophore and picks up a small portion of the emission spectra (usually including the peak emission). With this setup, only a fraction of the fluorophore emission spectra is recorded.

In spectral cytometry, we are no longer limited by the one detector=one fluorophore setup. Instead, all the detectors in the cytometer capture a signature of the full emission spectra of each fluorophore. The raw data is mathematically unmixed to identify the fluorophores. This allows for the discrimination of similar fluorophores, as long as they have unique signatures. For example, Alexa Fluor 647 and APC and be spectrally unmixed.

  • We have a 5-laser Aurora with the ultraviolet, violet, blue, yellow-green, and red lasers.
  • The number of colors that can be run on the Aurora is currently limited by the mathematics of the spectral unmixing and the number of fluorophores available. Mathematically you can unmix the number of detectors minus 2, so for our 64-detector Aurora we can unmix 62 fluorophores. Cytek has done all of the work to determine 30 fluorophores that can be used in one panel, so right now we can definitely run 30-marker panels. If you would like to run a larger panel, we just need to do some troubleshooting to figure out what additional fluorophores can be used.
  • As far as fluorophores, the Aurora can detect anything fluorescent – brilliant violets, alexa fluors, super brights, Qdots, viability stains, etc.
  • The aurora also has a volumetric counter for cell counts and a 96-well plate loader.
  • One thing that may be challenging to run on the Aurora is a cell cycle stain (like DAPI) in addition to other fluorophores. DAPI can still be used for live/dead discrimination.
  • Your samples can be prepared and stained exactly the same as you would with conventional cytometry
  • You are welcome to try any panels you have made for the conventional cytometers and see how they look (it’s a great way to test out the Aurora!), however it is recommended that you pay close attention to panel design. The larger the panel, the more important panel design becomes.
  • The software on the Aurora is similar to FACS DIVA, so you should be able to pick it up quickly
  • Setup on the Aurora is easier because you typically don’t have to change voltages/gains like you do on the conventional flow cytometers
  • Because the spectral unmixing is done mathematically and cannot be done manually, the Aurora is very particular about the single stains (called reference controls) being correct. See the training materials below for more information on reference controls.
  • Currently, spectral unmixing can be done with the Cytek SpectroFlo software and in FlowJo, but once it is completed, you can analyze your data with any program that takes .FCS files (FlowJo, FCSExpress, etc.). SpectroFlo is much easier to use than FlowJo for unmixing, so we recommend using SpectroFlo on the analysis workstations.

For training, please contact Laura Johnston.



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