The University of Iowa Carver College of Medicine

Flow Cytometry Facility

Sorting with the BD FACS Aria II
or BD FACS Aria Fusion

Cloning vs Bulk Sorting

Subpopulations of cell suspensions can be physically separated by the FACS Aria II and FACS Aria Fusion cell sorters into a variety of sort recepticles for cloning or bulk sorting. Both cell sorters can place a single cell in each well of a 6, 24, 48, 96, or 384-well plate for the purpose of cloning. The cell sorters can also sort any number of cells into each well of a 6, 24, 48, 96, or 384-well plate, 1.5 ml Eppondorf tubes, 12 x 75 mm test tubes, or 15 ml centrifuge tubes for the purpose of bulk sorting. When sorting into 6, 24, 48, 96, or 384-well plates, only one subpopulation can be sorted per well at a time, while up to four populations can be sorted simultanously for bulk sorting. (see later section on this page for detailed sort receptacle information)

Biohazard Sorting

Policy for Sorting Live Human Cells, Infectious Agents, Cells Infected with Replication Incompetent Virus, or Cells from Animals Previously Exposed to Infectious Agent

Sample Tubes

Cell suspensions can be placed in 1.0 - 2.0 ml cryotubes, 12 x 75 mm test tubes, or 15 ml centrifuge tubes for use with the FACS Aria II or FACS Aria Fusion. Biohazardous samples should be placed in tubes with caps.

Cell Concentration

The final cell concentration for cell sorting should be between 5 x 106 and 30 x 106 cells per ml depending on whether the cells are resting lymphocytes, activated lymphocytes, cell lines, disaggregated solid tissue, or fibroblasts. A list of primary cell types and their associated nozzle size can be seen within the online reservation system at the "Aria / Fusion Cell Types" link.

  • Recommended Cell Concentrations for Cell Sorting
          Cell Type    Cell Concentration Nozzle Size / Sheath Pressure       Max Sort Rate
    Resting Lymphocytes          20-30 x 106 / ml       70 um / 70 psi              22,000/sec
    Activated Lymphocytes          10-20 x 106 / ml       85 um / 45 psi              11,000/sec
    Cell Lines            5-10 x 106 / ml     100 um / 20 psi                9,000/sec
    Disaggregated Solid    Tissue            5-10 x 106 / ml     100 um / 20 psi                9,000/sec
    Fibroblasts               5 x 106 / ml     130 um / 10 psi                2,500/sec

Cell Sample Filtering

Just prior to sorting, cells should be filtered through nylon mesh. 70µm mesh filters (Falcon 2350) available through Biochem Stores are recommended. Filtering cells greatly reduces the probability of plugging the instrument during sorting. As a general rule, we will not sort unfiltered samples. We want to ensure a successful sort and once the instrument is plugged, it may take a significant amount of time to bring the instrument back to its original configuration. Time used to unplug the nozzle and bring the instrument back to sorting status may use up your scheduled time. If the cell preparation tends to re-clump after filtering, then addition of DNase to the sample may help eliminate aggregation. See Disaggregation of Clumps Using DNase for more information.

Sort Collection Media

Improved post-sort cell viability can be acomplished by keeping the sort collection media pH constant and providing a source of protein to the sorted cells. Normal cell culture media uses a CO2 buffering system normally supplied by an incubator. Exposing this media to air during cell sorting allows the pH to drift. It is therefore recommended that PBS or Hepes buffered culture media be placed in the sort collection vessels plus enough serum to replicate culture conditions. The final fluid volume in the sort collection vessel will be a mix of collection media plus cell sorter sheath fluid deposited as result of sorting. The amount of serum should reflect the final expected volume.

Sort Collection Receptacles

The FACS Aria and FACS Fusion can sort into 1 ml microtubes, 1.5 ml Eppendorf tubes, 12 x 75 mm test tubes, 15 ml conical centrifuge tubes, 96-well or 384-well plates. If the sorted population consitutes from 10% to 99% of the original population, 15 ml conical centrifuge tubes should be used. The tubes should be filled with 5 ml of sort collection media (see information in previous section of this page). If the sorted population is less than 10% of the original, then the 15 ml collection tubes should be filled with 10-13 ml of media or 12 x 75 tubes used with several milliliters of media. If sorting into 96-well plates, 100-200 ul (200 ul recommended) of media should be placed in each well prior to sorting.

Spinning the plates post sorting for 30-60 seconds at 300xG will help cells adhere to the plate and increase the number of colonies that will grow.

  • Cell Sorting Collection Receptacles
  • Cloning (one cell per well)
  • 96 or 384-well plate

  • Bulk Sorting
  • 1.5 ml Eppondorf tubes (two-way or four-way sorting)

    12 x 75 mm test tubes (two-way or four-way sorting or three 12 x 75 tubes plus one 15 ml tube)

    15 ml centrifuge tubes (two-way sorting or three 12 x 75 tubes plus one 15 ml tube)

Cell Sorter Sheath Fluid

Sorted cells ride in droplets composed of sheath fluid on their way to the sort collection tube. Once the cells have arrived in the collection vessel, they are mixed with the sheath fluid from the droplets and culture media that has been placed in the collection tube. There are 3 choices of sheath fluid that can be used in the FACS Aria or FACS Fusion:

  • Facility supplied with antifungal/antibacterial agent
  • Facility supplied without antifungal/antibacterial agent
  • 1X PBS (supplied by investigator)

Both Facility supplied sheath fluids are essentially PBS with or without an antifungal/antibacterial preservative agent (Proclin 300). Most cell types tolerate exposure to the sheath fluid preservative and thrive after sorting. Some cells, such as human stem cells and human dendritic cells, do not tolerate exposure and tend to die quickly. In experiments where cells may not tolerate exposure to the sheath fluid preservative, we recommend substituting either Facility supplied preservative-free sheath fluid or 1X PBS. To allow enough set up time to prepare the instrument using 1X PBS, the lab requesting the sort should bring 4-10L for the FACS Aria or FACS Fusion to the Facility the day before the sort. The amount needed for the sort will depend on the length of time scheduled. Please ask one of the Facility personel for advice on the amount of PBS needed.

Phenyl Red

Resuspension of cells to be analyzed in media containing phenyl red should be avoided whenever possible. Phenyl red may increase the background fluorescence of cells.