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Sample Preparation for Sorting

Sample preparation for using the Becton Dickinson FACS Aria II or FACS Fusion cell sorters.

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)

Excyte Expert Cytometry media recommendations for suspending cells to be sorted.

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.

Amount of Cell Sorter Time to Reserve (Cells per Hour)

          Cell Type    Cell Concentration      Nozzle Size /  Sheath Pressure            Cells/Hour
    Resting Lymphocytes          20-30 x 106 / ml       70 um / 70 psi              60x10^6/hr
    Activated Lymphocytes          10-20 x 106 / ml       85 um / 45 psi              30x10^6/hr
    Cell Lines            5-10 x 106 / ml     100 um / 20 psi                22x10^6/hr
    Disaggregated Solid Tissue            5-10 x 106 / ml     100 um / 20 psi                22x10^6/hr
    Fibroblasts               5 x 106 / ml     130 um / 10 psi                7.5x10^6/hr

Recommended Cell Concentrations for Cell Sorting

          Cell Type    Cell Concentration      Nozzle Size /  Sheath Pressure            Max 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 or other agents to the sample may help eliminate aggregation. See Disaggregation of Clumped Cells 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 II 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 96-well 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.

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.

Sorting Fixed Cells for RNA Sequencing

Preserving high quality RNA for post-cell-sort sequencing in fixed cells can be achieved using a zinc-buffer fixation protocol.

Information posted March 27, 2019 on the Purdue-administered flow cytometry bulletin board by Dr. Roxana del Rio-Guerra says -

"We, the (ABRF-) Flow Cytometry Research Group just presented our work at the last ABRF meeting (poster 129) about the different fixative methods (including commercially available products for that purpose) and the purity, quality and yield of RNA after sorting cells. We tested: paraformaldehyde (2 and 4%), formaldehyde (1 and 4 %), alcohols (ethanol and methanol), Zinc buffer, as well as commercially available kits such as CytoFix/Cyto perm and FACSLyse (from BD), and IC fix/perm buffer (eBio/ThermoFis). Each of these protocols was performed by two different Flow core labs at two different institutions and all samples sent to a general lab for evaluation of the integrity/quantity of RNA. Among all those protocols, the Zinc Buffer protocol (Christensen, R, et al. Curr Protoc Cytom 2011, Chapter 7: Unit 7.40. PMID: 21732310) gave us a very promising results with near to 8.0 on the RNA integrity number. For our work, we first stained cells only with a viability dye and then fix-sort-fix."

In a follow-up message Dr. del Rio-Guerra adds -

"Also, on ref from Curr Prot Cyt, we changed from Zinc Acetate to 17.16 mM zinc-trifluoroacetate since this was shown to better preserve the quality of RNA (ref PMID: 17576663) We tested: after fix-perm (with the diff fixatives mentioned), sorted cells were collected on RLT plus buffer (Qiagen 1053393). With the exception of Zn buffer, quality of RNA was very poor. The results presented at the last ABRF meeting are preliminary, but the Zn buffer protocol was the one reproducible in two different core labs and with the best quality of RNA. We will continue working on this project."

If you have access to Current Protocols in Cytometry , you can access the zinc-buffer fixation protocol directly. The Flow Cytometry Facility can provide the protocol in PDF on request.

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.