University of Southern California

Sorting FAQs

Q: How long should I make my appointment for?

A: Generally speaking, the cell sorter will aspirate 1 mL of volume in 30 minutes. This assumes there are no delays from nozzle clogs and the flow rate of 4.0 is used. For multiple arm experiments, additional time is required for cleaning between samples to minimize carryover. Additional time is needed to acquire the controls and perform compensation.

Q: How many cells should I bring?

A: The number of cells to prepare for flow cytometry experiments can vary drastically and depends heavily upon the detection assay and purpose of the experiment. Beginning with a surplus of cells is ideal; however, source size may be the limiting factor. For cell analysis, beginning with 1 x 10^6 cells is always a good place to start. For cell sorting, the cell number often becomes 1 x 10^7 or more.

Q: How many cells will I get back?

A: The number of cells returned depend upon the following factors: starting cell number, sort precision, sort efficiency and target frequency. Users can assume that the sorter will loose 50% of the possible targets to find the “worse case” return.

Q: Which sizes of tubes work in the cytometers?

A: The Aria I and Aria II cell sorters can accept the following tube formats: 1 mL tube, 1.7 mL Eppendorf with lid removed, 12×75 mm polystyrene or polypropylene tubes, and 15 mL conical centrifuge tubes. For sort receiving (AKA collection) tubes, the Arias can accommodate the same tube formats. The MoFlo can accommodate the following tube formats: 1 mL tube, 1.7 mL Eppendorf with lid, 12x75mm polystyrene or polypropylene tubes, and 15mL or 50mL conical centrifuge tubes.

Q: Which controls do I need?

A: The USC Flow Cytometry Core recommends the use of unlabeled cells, single stained tubes and FMO controls to properly set up flow cytometry experiments. These are required in addition to the experimental controls your project requires. DNA cell cycle projects to not require unlabeled cells. Questions regarding experimental design should be discussed with core personnel prior to experimentation.

Q: Does the lab run Quality Control?

A: Yes. CS&T quality control assay is performed on the BD Biosciences instrumentation and Ultra-Rainbow beads are tracked on the Beckman Coulter instrumentation.

Q: Which nozzle size should I request?

A: Most adherent cell lines and tissue dissociations use the 100 um nozzle, while most suspension cells use the 70 um nozzle. There do exist sensitive cells that may require other specialized nozzles. Discuss with the director if you are unsure what to request.

Q: Which concentration of cells is best for cell sorting?

A: Users should count their cells using a hematcytometer or some other volumetric means before diluting to a final concentration. Adherent cells can be concentrated up to 20 x 10^6 cells per mL. Suspension cells can be concentrated up to 100 x 10^6 cells per mL. We recommend users bring diluent with them to adjust the cell concentration as necessary to achieve maximum sort rate.

Q: What is the maximum sort rate?

A: The maximum event rate for purification on the sorters should not surpass ¼ of the drop drive frequency. High speed sorting creates 85,000–100,000 droplets per second.   Low speed sorting creates 30,000–50,000 drops per second. Special setups using atypical nozzles will deviate from these settings. In all cases, the cell concentration is adjusted to maintain sort efficiencies of 75% or better. We recommend diluent is brought to the appointment.

Q: What does “sort precision mode” mean?

A: Sort precision determines how the cell sorter sets priorities to collect target cells. If set for Purity, the sorter will exclude droplets containing unwanted cells as well as throw away cells nearby contaminants. When sorting for purity, the sorter will have reduced recovery. When set to Enrich, the sorter will sort every possible target without regard to purity. If count accuracy is desired, the sorter will place emphasis on cell count and purity. This sort precision is called “single” and will sacrifice the most target cells to achieve both count and purity demands.

Q: Why are the sorters in biosafety enclosures?

A: The cell sorters are commonly called “Jet-in-Air” cell sorters. The sample is introduced to the instrument under pressure, passes through the lasers and then exits the system via a vibrating nozzle orifice at high pressures. These electrostatic sorting systems therefore create aerosols to perform the work. The placement of the cell sorters in the biosafety cabinet reduces the risk assessment associated with sorting human cells using electrostatic means.

Q: Does the core accommodate emergency sorting?

A: The core director decides if emergency cell sorting can be performed.

Q: Which sheath buffer is used in the cell sorters?

A: The cell sorters use proprietary sheath buffers suggested by the manufacturers.   FACSFlow is used in the Aria cell sorters and IsoFlow is used in the MoFlo. Both reagents are phosphate-base, contain EDTA as well as bacteriostatic agents.

Q: Why should I stain for dead cells?

A: Staining your sample for dead cells will improve the quality of sorted sample. If dead cells are labeled and removed from the sorted population, the user will end up with more viable cells to perform downstream experimentation.

Q: Are there BSC and incubators available in the core?

A: Yes. The USC Stem Cell Core Facility maintains a communal space complete with BSC and incubator access. USC Flow Cytometry Core clients may use Biosafety Cabinet #6 and Incubator #14 found in Tissue Culture Room 209B. Users are expected to adhere to posted usage guidelines.

Q: What are good collection medias?

A: Optimal collection media for cell culture will contain HEPES and high serum. Cells will be delivered to this media and mixed during/after the sort process. Cells should be centrifuged out of this media prior to re-culture. Alternatively, users may sort directly into RNA stabilizer reagents that do not contain phenol. Phenol is not permitted in the USC Flow Cytometry Core; the biosafety cabinets are not rated as chemical hoods.