Choosing a Sorter

The FCR houses two cell sorters for investigators to choose from.

The BD Symphony S6, Agnes, is a traditional sorter with 5 lasers capable of managing panels approaching 24 colors.  This instrument is an updated version of the Aria with hardware and software continuity

Our Cytek Aurora CS, Link, is a full spectrum sorter with 5 lasers, capable of managing panels approaching 40 colors.  Link is also matched to both of the Aurora analyzers, Constantine and Sweetums, so this will allow you to transfer files/experiments between all three instruments. 

There is much parity between the S6 and the CS and it is likely that either instrument will serve an investigator’s needs adequately. The largest distinction between the two is the difference between conventional and spectral cytometers. Bear that in mind when choosing which platform with which to work. Because of the way spectral unmixing works and due to their ability to extract autofluorescence, spectral sorters can be a bit more demanding when it comes to single stained and unstained controls, however this is greatly to the benefit of the investigator as autofluorescence extraction can improve the resolution of fluorescent signal. Additionally, if an investigator has already done work on the full spectrum analyzers, entire experiments or elements of experiments (such as gating schemes or reference controls) can be transferred directly into the software on the CS to streamline experiment setup. 

You can review each sorter’s configuration page (linked above) and their specific capabilities in Table 1 before choosing an instrument. 

If you have any questions or concerns about choosing a sorter that’s right for you, please contact a staff member so we can address your needs

TABLE 1 – Instrument Capabilities Table

 SAMPLE TUBES (See TABLE 1)

• Both sorters can accept samples (to sort) in 1.5mL, 5mL, or 15mL tube sizes.
• BD brand tubes typically work best.

COLLECTION VESSELS (See Table 1)

COLLECTION TUBES

• Both sorters can collect into 1.5mL, 5mL, 15mL tubes.  
• The BD S6 can also collect into 50mL conicals 
• The sorters can collect up to 6 sorted populations depending on collection vessel.  You can find the specific MAX block configurations for each instrument in TABLE 1.

COLLECTION PLATES AND SLIDES

• Sorting bulk amounts or single cells can be accomplished for plates and slides.
• Both sorters can sort into plates, from 6-well to 384-well, as well as onto slides.
  1. If using a plate manufactured by a company other than BD, Corning, or Costar, please contact FCR staff so we can ensure the form factor of your plate will fit.
  2. Contact FCR staff for more information 

COLLECTION MEDIA

• The suggested minimum appropriate volume of media for a collection tube is 10% of the volume of the tube (i.e. 500uL in a 5mL tube), however that can be adjusted if necessary
  1. To aid in the recovery of a rare sort population of low percentage in the sample, a high volume of media in a conically shaped collection tube is recommended (750uL in a 1.5mL Eppendorf).
• Media in collection tubes should be chosen based on the needs of the sorted cells.  Sorting for continuing culture will be very different than sorting for downstream genomics work.  There are no specific requirements from the FCR for collection media. 
• It is important to pre-coat collection tubes with media containing serum to increase post-sort viability and recovery. This involves coating the entire inner surface of the collection tube. Sorted cells that hit bare plastic have the potential to stick and dry out or burst.

• If an investigator intends to sort into lysis buffer they should contact the Flow Core staff before their sort to discuss dilution restrictions of the buffer and determine a reasonable maximum amount of events to collect in each collection tube. 

COLLECTION VESSEL CAPACITY

• Collection Tubes can hold (approximately) the following number of cells based on the nozzle used. These numbers are based on collection tubes that have 10% of the volume as collection media.
• The approximate droplet volume for the standard 85um nozzle configuration is 2.5nL.

 TABLE 2 – Tube Capacity Table

The figures in the above table can be influenced by the sort precision mode that is chosen as well as the frequency and amplitude with which droplets are being generated (on the S6 only, which changes droplet volume.) If an Investigator thinks modifying the droplet size will be beneficial to their sort or simply wishes to know more about the method they should inquire with the Flow Core staff. 

Nozzle Size

• The largest determining factor in selecting a nozzle size is the size of your cells. 
  1. As a general rule, your nozzle should be at least 2-3x larger than the largest cell type that will be present in the sample you are going to sort. 
• The fragility of the cells to be sorted is another significant factor. Fragile cells would benefit from the lower pressures of the larger nozzle sizes.  See Fluidics Pressure Table for details. 
• We encourage investigators unsure of which nozzle size to select to provide a small sample of their cells a before the proposed sort to allow us to help determine which nozzle to use.

• Nozzle Options

1. The default configuration of the sorters use the 85 micron nozzle. We have found that this nozzle configuration works very well for most of the cell types we sort – from lymphocytes to a multitude of cell lines. 
2. If you have a larger cell type or need a gentler sort we can switch to a 100 micron nozzle.  
3. There is also a 130 micron nozzle but it is rarely used and only necessary for very large cells or very low pressure sorts.  Switching to the 130 micron nozzle can take an additional 30 minutes in order for the fluid stream to stabilize so be sure to build that into sort time calculations.
4. Smaller cell types or bacteria can be sorted using the 70 micron nozzle.

 TABLE 5 – Fluidic Pressure Table 

Sort Precision Modes/Sort Masks

Our sorters have various Precision Modes to choose from, which will affect the way the machine sorts cells.

TABLE 3 – Sort Precision Mode Table