Choosing a Sorter

The HMS Department of Immunology flow core offers two high speed cell sorters. We have a 4 Laser BD FACSAria II and a 6 Laser Beckman Coulter MoFlo Astrios EQ. Each sorter has its own set of strengths and weaknesses and associated colors that can be detected. It is important to consider the attributes of both pieces of equipment when deciding which sorter to book for your experiment.

Laser Interrogation:

The largest difference between the sorters lies in the location of laser interaction with the cells. The FACSAria uses a quartz cuvette for interrogation and fluorescence excitation while the MoFlo uses a jet-in-air system. The cuvette based interrogation on the Aria provides better optical resolution for distinguishing dim populations from background fluorescence and has better scatter resolution for gating out dead and aggregated cells. This increased optical resolution comes at a price though. Cells are slowed down when they pass through the lasers and sped back up before ejection. These changes in speed cause sheer forces that can be stressful on the cells. The jet-in-air approach of the MoFlo maintains a steady cell velocity minimizing stress on the cells and improving cell viability after sorting.

Lasers and detectors:

While both sorters are capable of detecting all the common dyes, they do not contain the same set of lasers and detectors. The Aria is a 4 laser, 15 color system while the MoFlo has 6 lasers and 25 fluorescence detectors. Please visit the equipment page to view the optical configurations of each instrument. The most notable difference is the UV laser found exclusively on the Aria and the 561nm yellow/green laser found only on the MoFlo. The Aria’s UV capabilities allow for the detection of DAPI or Hoechst for live/dead discrimination while keeping all other channels open. The 561 laser on the MoFlo is necessary for mCherry excitation and opens the door for more color choices while minimizing spectral overlap.

Sort collection devices:

Both sorters can bulk sort several populations simultaneously or sort single cells into plates for culture or single cell analysis. The Aria is limited to sorting 4 populations at once while the MoFlo is capable of sorting up to 6 discreet cell populations. The MoFlo also has more reliable single cell deposition allowing for reliable 384 well plate sorting while the Aria is limited to only sorting into 96 well plates.

 

Sort Guidelines
Sort Sample Requirements:
  • Cell concentration not to exceed 107 cells/mL
  • Cells are to be filtered through 45 uM mesh just prior to sorting (after final wash or staining step)
    • BD filter cap tubes item #352235 work well
  • Cells can be brought in 12x75 mm round bottom tubes or 15 mL conical Tubes
  • Recommended cell suspension media: 1-3% serum and 2mM EDTA in DMEM or PBS. Excess protein (BSA, FBS etc) causes background scatter, auto-fluorescence, and promotes clogging
  • Phenol red is not recommended in cell suspension media
Controls Requirements:
  • An unstained control and a single positive control for each fluorophore used is required
  • Controls should be brought in a volume of 0.5-1mL and preferably contain approximately 106 cells
Collection Tube Requirements:

Tube Sorting:

  • All collection tubes should be filled approximately ¼ with desired buffer (cell growth media, PBS, lysis buffer etc)
  • Eppendorf tubes or 12x75 mm round bottom tubes can be used for up to 4 way sorting on the Aria and 6 way sorting on the MoFlo Astrios
  • 15 mL conical tubes can be used for up to 2 way sorting (both sorters)

Plate Sorting:

  • BD FACSAria: Cells can be sorted directly into 6, 12, 24, 48, or 96 well plates
  • MoFlo Astrios: : Cells can be sorted directly into 6, 24, or 96 or 384 well plates
  • Wells should be brought pre-filled with an appropriate buffer
  • Please indicate plate type on the calendar at the time of sign up
  • If a non-standard plate is being used (such as a flexible V-well PCR plate) please bring an empty plate to the core at least 1 day prior to your sort