SAMPLE CONCENTRATION

• Sample concentration can be from 10x10e6 events/mL to 35x10e6 events/mL depending on nozzle size.  See the Table 4 Sample Concentration and Sort Rates for details.
  • “Events” is used instead of “cells” to denote concentration because the sorters do not differentiate between cells and debris in the sample with respect to what is detected and displayed.

TABLE 4 – Sample Concentration and Sort Rates

SAMPLE TRANSPORT

• Transport of BSL-2 samples through the University must be done in a secondary container that has a secure lid. The outer container must have biohazard labels on it. Transportation in Styrofoam boxes, for example, is unacceptable.
• Since the FCR staff or anyone you might encounter, does not know if you have BSL-2 level material, ALL samples coming to the flow core must be transported in secondary containment.
• Failure to transport samples safely will result in a warning, and repeated offences will result in access to the FCR being revoked until a review of appropriate procedures with the FCR director is completed.

SAMPLE PREPARATION

• FILTERING

  • Samples must be filtered, preferably using a 35uM filter if possible, prior to being sorted.

• ADHERENT CELLS

• Adherent cells pose a special problem for a sorter. In general, these cells have been treated with trypsin to remove them from the culture plate. Standard practice is to then add serum to neutralize the trypsin, however, this causes problems as trypsin adds back divalent cations necessary for cells to adhere to each other. **It is better to use a trypsin inhibitor to inactivate your trypsin rather than serum**. 
• Adherent cells, activated cells and cells with extensive structures outside the cell membrane all benefit from a slower sort (lower sample concentration, lower flow rate).

• DNase 

  • As cells die they can release their DNA into the sample buffer. This released DNA readily binds to other cells causing clumping and reducing the effectiveness and quality of the sort. 
  • The addition of 10 Units of DNAse per mL of sample will help prevent this effect. This is especially important with adherent cells.

Proper Controls 

Adequate controls and supplies help ensure a quality sort.

1. Unstained Cells/Negative control – so we can establish the size and fluorescent background of your cells.
2. Single Stained Controls – For each fluorochrome you are using.  These can be either beads (“Simply Cellular Compensation Standard” beads from Bangs Lab, “BD CompBeads” from BD Biosciences, etc.) or cells. It is important that you have a positive and negative in your control samples.  Likewise, if you use beads for compensation, please make sure to provide a negative (unstained) bead as well. **If you have questions about compensation, compensation controls, or experimental controls, please contact the Flow Core staff for assistance!** 
3. A known positive control is useful for reference if possible.

What to Bring with You for Your Sorting Experiment:

** Transport of BSL-2 samples through the University must be done in a secondary container that has a secure lid. The outer container must have biohazard labels on it. Transportation in Styrofoam boxes, for example, is unacceptable**

1. Unstained Cells and/or Negative Control 
2. Single Stained Controls 
3. Positive Control 
4. FMO’s, if applicable
5. Samples at an appropriate concentration for the nozzle being used
6. Collection vessels for sort with media
7. Extra Collection vessels
8. Extra buffer with which to dilute your sample if necessary
9. Extra media to make up new collection tubes if necessary
10. Sort Logic