University of Southern California

Analysis FAQs

Q: On the LSR, what is the difference between the three speeds?

A: LO is 12 uL/min, MED is 35 uL/min, and HIGH is 60 uL/min at the half-way point on the variable adjust knob. The variable adjust knob takes the sampling volume from 0.5X (fully counter-clockwise) to 2X (fully clockwise). The LSRII should not be considered a “cell counter” without proper calibrating beads, such as Tru-Count beads.

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 flow cytometric 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. You will sample some of these for your data set. Rare event analysis can only be achieved if large samples are taken and measured.

Q: Which sizes of tubes work in the cytometer?

A: The Cyan and LSRII machines accept the FALCON brand 12×75 mm polystyrene tubes. The Cyan can also accept 12 x 75 mm polypropylene 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 in addition to experimental controls for most immunophenotyping projects. 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: What biosafety training is required?

A: USC employees, undergraduates, graduate students and postdocs are expected to have General Laboratory Safety and Blood Borne Pathogens training as given by USC-EHS. The EHS training schedule can be found at: capsnet.usc.edu/department/environmental-health-safety/environmental-health-and-safety-training.

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.