The general consensus is that viability, not unlike life itself, is pressure
sensitive: some can take it better than others.
All agree that viability of murine lymphoid and bone marrow cells is
excellent when sorted at 30 to 33 psi with the BD TurboSort. Functionality,
and colony forming potential of progenitors is uncompromised. One brave
soul found that at 45 psi, using a 70 micron tip, only 6% of the lymphoid
population and 8% of the granulocyte population were PI positive after Turbo
sorting.
With respect to human myeloma cell lines there was no reported effect on
viability, and only a small difference in colony forming units in number and
size. Turbo sorted(human) CD34 positive cells had the same viability and
colony forming potential as non-Turbo sorted cells.
There were a few responses from the MoFlo camp: viability at 60 psi is
excellent, with only 2% of the population staining PI positive after
sorting, and 7% staining PI positive at 80 psi (mouse bone marrow). One
respondent indicated that using the MoFlo, single bacteria cloned out at
100%, and bull semen gave pregnancy rates similar to unsorted material. Ok ....
The exceptions are fragile cells: ie destined for apoptosis heaven [ should
use less than 22 psi ], or stimulated (spenocytes) [22 psi ], dendritic and
microglial cells [12 psi]. These cell types benefit from lower sort
pressures and larger nozzle tips.
Selection of nozzle tip diameter is important if not critical for good
results: large, more fragile cells show less mortality with larger diameter
tips. Nozzle tip design ... I have to assume the MoFlo uses a different
design from BD ... also influences viability at increased pressures (kinda
intuitive, but the implication is that nozzle tip selection has more effect
on viability than pressure for most cell types).
There is the question of TurboSort use with the autoclone unit: Why do this
at high speed since especially in 96 well plates, far more cells will go to
the waste as the unit travels between wells. There may be some gain in
sorting for very rare events or sorting large numbers of cells into 24 well
plates however.
And finally, last but certainly not least, many agree that the biggest
factor in cell viability ... in most cases ... is cell prep, not sorter
pressure. Recommendations include the use of HEPES buffered saline over
phosphate buffered saline since the former is less susceptible to changes pH
with exposure to the atmosphere. Verifying the osmolarity of the sheath
fluid is also recommended.
And that about sums it up.
PS: don't ask me, I don't have a high speed sorter ... maybe some day I
will, judging from the responses in this survey.
Again, sincere thanks to all who responded,
Claude