The University of Iowa Carver College of Medicine
Sorting with the BD FACS Aria
Preparing cell samples for sorting with the Becton Dickinson FACS Aria.
Cell suspensions can be placed in 1.0ml microtubes, 12 x 75mm test tubes, or 15ml centrifuge tubes for use with the FACS Aria. Biohazardous samples should be placed in tubes with caps.
The final cell concentration for cell sorting should be between 5 x 106 and 30 x 106 cells per ml depending on the concentration that the cells tend to aggregate. For lymphocytes, the suggested concentration is 20-30 x 106 per ml. Cell line concentration should be 5-10 x 106 per ml. As a rough estimate, for every 10 million cells per ml, the instrument can be run at 10,000 cells per second. For example, if lymphocytes were concentrated to 20 million per ml, the flow rate at the instrument could be run as high as 20,000 cells per second. Cells larger than lymphocytes require a larger nozzle to be placed on the instrument and the flow rate is lower.
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 insure 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 Vessels
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. Where the sorted population consitutes from 10% to 99% of the original population, 15 ml conical centrifuge tubes should be used. They 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 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.
Cell Sorting Rates
|Nozzle Size||Sheath Pressure||Sort Rate|
|70 um||70 psi||22,000/sec|
|85 um||45 psi||11,000/sec|
|100 um||20 psi||9,000/sec|
|130 um||10 psi||2,500/sec|
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.