The University of Iowa Carver College of Medicine

Flow Cytometry Facility

Sorting with the BD FACS DiVa

Preparing cell samples for sorting with the Becton Dickinson FACS DiVa.

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 should be placed in Falcon brand (352052 or 352054 {with caps}) polystyrene 12 x 75mm test tubes for use with the FACS Diva. Biohazardous samples should be placed in tubes with caps. Biochem Stores carry the correct tubes.

Cell Concentration

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 352350) 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 as long as an hour 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 Tubes

The FACS DiVa can sort into Eppendorf tubes, 12 x 75mm test tubes, 15ml conical centrifuge tubes, or 96 well plates. For sorts where the sorted population constitutes from 10% to 99% of the original population, 15ml conical centrifuge tubes are recommended. They should be filled with 5ml of culture media to keep the sorted cells healthy. If the sorted population is less than 10% of the original then the 15ml collection tubes should be filled with 10-13ml of media or 12 x 75 tubes used with several ml of media.

Sort Media

There are three types of sheath fluid that can be used in the DiVa: Facility supplied with antifungal/antibacterial agent; Facility supplied without antifungal/antibacterial agent; and 1X PBS. 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 4-6L of 1X PBS 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 personnel for advice on the amount of PBS needed.

Phenyl Red

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