CMRC Children's Memorial Research Center
Protocols

Points to Consider Prior to Submitting Samples for Sorting

Cell Concentration
While a starting concentration for cells to be sorted is from 5 X 106 to 30 X 106 cells/ml, the actual concentration best for each sample will be highly dependent on the concentration at which the cells begin to aggregate. A suggested concentration for lymphocytes is 20-30 X 106 cells/ml while for different cell lines is 5-10 X 106 cells/ml.
 
Cell Sample Filtering
The last step in preparing cells for sorting should be to filter the cells through a 70 um mesh filter since filtering cells prior to sorting significantly reduces the probability that the FACSAria will become plugged during the sort. Mike Yu can provide sample filter units for a limited time for this purpose.
 
Sort Collection Vessels
The following vessels may be provided to the FACSAria facility for collection of the sorted samples:
  • 12 X 75 mm test tubes
  • 15 ml conical tubes
For consideration: If the sorted population constitutes from 10-99% of the original population, we suggest using 15 ml conical centrifuge tubes filled with 5 ml of culture media for collection and proper maintenance of the sorted cells. Sorted populations constituting less than 10% of the original sample should be collected into 15 ml tubes filled with 10-13 ml of medium or 12 X 75 mm tubes with 3-5 ml of medium.
 
Sort Media
Cells that are being sorted are carried in droplets composed of a sheath fluid. In general, the sheath fluid used for sorting live cells is PBS.
 
Avoid re-suspending cells in media that contains phenyl red since this could increase the background fluorescence of the cells.