Sorting and aggregates
Aggregates or clumps can severely affect the integrity of your sort no matter which instrument you work with.
Clumps cause clogs, which block or disturb the laminar flow process through the nozzle. Such disturbance usually results in redirection of the central core stream or spraying of the side streams, and usually affects the purity of the sort.
While we can manually manipulate the instrument to counteract some of the hazards of aggregates, three bench-based steps can help:
- Use PBS or HBSS without calcium and magnesium as the base of all the solutions you need in prepping cells for sorting. In some cases 1 mM EDTA is also recommended.
- Keep protein in your sort buffer to 0.5% (FCS or BSA). Collection vessels can have unlimited protein to keep cells happy once they’ve been sorted.
- Filter samples immediately prior to sorting by passing them through 50-70 um nylon mesh.
For primary tissue that has been homogenized or dissociated:
Cells need to be passed through a sieve to remove obvious chunks prior to staining but then, they should be filtered as described above just before the sort. The standard sorting nozzle size is 70 um.
For cultured cells, especially cells that don't like to be removed from a matrix or that tend to adhere:
In most cases, cells are sorted at slightly lower pressures and in more dilute concentrations. For very large cell types, standard sort nozzle size is increased to 100 um and the pressures and speeds are reduced even further. Regardless of the size of the cell, it must be filtered prior to sorting if it comes out of a culture dish.
