Cell Sorting

Help on how and why to sort your cells out of a heterogenous mixed population for a multitude of downstream analytical applications. 

The flow cytometry core facility has a FACS Aria III high-speed cell sorter capable of sorting cells at several thousands of events per second. The specific limitations and speed will depend on the cell type being sorted, sort stream size, pressures, etc.

This sorter is enclosed in a BSL-II hood, allowing sorting of limited human samples. Due to the fact that the sorter has a small particle detection unit, it can also assess particles of ~200nm and larger. The Aria is an 11 colour system with excitation sources of 405nm, 488nm and 633nm with higher fluorescence resolution due to a cuvette-based design and higher power 488nm laser.

All samples being sorted must be registered using a Facilities-Sorting-Sample-Registration in order to identify the biohazard risk level of the sample. Because sorters create bioaerosols, the biosafety level of your sample is not the same in a sorter as it is for other instrument use. Please download the form above and print it and fill it out. The form is quite short and if you need help filling it out, please contact the Flow Cytometry Specialist.

Cell sorting differs from “regular” cytometry due to the need to keep the cells alive after analyzing them and the sensitivity of cell sorters to aggregated cells. There are many sample prep and buffer recommendations, but the most basic are listed below:

  1. Filter the cell suspension through 50µm mesh immediately before sorting to remove large clumps that can clog the machine and compromise your entire sort.
  2. Use calcium and magnesium free buffer (PBS, HBSS, etc.) to prevent clump formation. In some cases 10mM HEPES or 1 mM EDTA can be included if necessary. EDTA will keep more sticky cells from re-associating.
  3. Use 0.5% BSA if possible (instead of the 1-2% standard for cell analysis). If you want more serum protein you can do so but they have to get there without clogging the sorter. It is common to include up to 50% FBS in your collection tube to keep the cells “happy” after they have been sorted.

The flow cytometry facility staff will be happy to assist you in setting up and optimizing your cell sort. Please realize that this often involves making sure that your analysis is optimized on a flow cytometer prior to sorting.

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