The University of Iowa Carver College of Medicine

Suggestions for Success

Cell Sample Preparation for Sorting with the Becton Dickinson FACS DiVa

Sample Tubes

Becton Dickinson FACS DiVa
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. Biochemistry Stores carry the correct tubes.
Becton Dickinson FACScan, FACS Calibur, LSR
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. Biochemisty Stores carry the correct tubes.

Cell Concentration

Analyzing
The final cell concentrations should be 1 x 10⁶ cells per ml for phenotyping, apoptosis, DNA content, GFP or similar types of experiments.
Sorting with FACS DiVa
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 slightly lower.

Sample Volume

Becton Dickinson FACS DiVa
For initial setup of the instrument a minimum of 250µl is needed at a concentration of 1 x 10⁶ cells per ml. After initial setup, the minimum volume can be reduced to 100µl at the previous cell concentration. The minimum recommended volume is 250µl.
Becton Dickinson FACScan, FACS Calibur, LSR
The minimum volume that can be run is about 300µl. The minimum recommended volume is 500µl at a concentration of 1 x 10⁶ cells per ml.

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 consitutes from 10% to 99% of the original population, 15ml conical centrifuge tubes should be used. They should be filled with 5ml of culture media. If the sorted population is less than 10% of the original then the 15ml collection tubes should be filled with 10-13 ml of media or 12 x 75 tubes used with several ml of media.

Sort Media

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 DiVa:

Facility supplied with antifungal/antibacterial agent
Facility supplied without antifungal/antibacterial agent
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 personel for advice on the amount of PBS needed.

Compensation Controls for Multi-fluorochrome Experiments

Flow Cytometry technology can yield highly accurate data, but only if the proper controls are done prior to sample analysis. This is especially important when multi-color analysis is undertaken. Spectral overlap between fluorochromes in multi-color experiments requires the use of compensation. (See http://www.drmr.com/compensation/index.html for an in depth explanation of compensation.) Without proper compensation, the data obtained would be meaningless. The proper compensation controls may vary from experiment to experiment, but some basics are the same. Thus, an unstained or isotype control and a positive control for each color are essential.

Example: For mouse splenocytes stained with FITC, PE, and PE-Cy7 conjugated antibodies, compensation controls should include:

tube 1) unstained or isotype stained for each color
tube 2) anti-CD4 FITC
tube 3) anti-CD8 PE
tube 4) anti-CD8 PE-Cy7

Experiments that cannot spare cells for compensation controls can use a bead product from Becton Dickinson called BD CompBeads. The beads can be stained with antibody specific for the Kappa light chain for mouse, rat, or rat/hamster immunoglobulins. The beads are stained as if they were cells using the same antibodies and fluorochromes that are used in the experiment. In this way, a set of compensation tubes can be produced using beads instead of cells that have both a negative and bright positive population for each color. If the beads are used, the experiment would only require the addition of an unstained or a negative tube of cells to set the initial baselines.

The Facility's mission is to serve the investigators in their quest to obtain accurate data. The lack of proper compensation may yield misleading, confusing and inaccurate data. In order to live up to our mission of assuring quality control and reproducibility, the Facility will not run the samples if the necessary compensation controls are not provided by the investigator.

Aggregates

Certain cell types like monocytes, granulocytes, and adherent cell lines tend to be sticky and form aggregates. These aggregates will plug the instrument (a 77um aggregate will not go through a 76um jet). If you can "see" anything in your sample tube, it probably means that the cells have aggregated. Those samples must be filtered with nylon mesh to remove the aggregates or dispersed by some other method before running on the flow cytometer. It has been reported that EDTA and DNAase (used on unfixed cells) will help prevent aggregates. A commercial product, Accumax, has been developed for the specific purpose of keeping cells from clumping. Other sources of large debris such as solid tissue should also be filtered with nylon mesh. In general, anything that you can "see" in the sample tube is too big to go through the instrument.

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.

Phenyl Red

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

Fixing Cells with Formaldehyde and Increased Autofluorescence

When fixing cells for immunofluorescent experiments with formaldehyde a common problem is increased autofluorescence. The resultant decrease in separation between the negative and positive populations can render some experiments useless. The most common reason for increased autofluorescence is pH drift of the formaldehyde. It is important that correct pH is established in fresh formaldehyde and that pH is monitored as the fixative solution ages.