Sorting HUH cells... poor viability, clumping

Miller, Mark A. mark_miller2 at Merck.com
Mon Sep 11 13:27:57 EST 2000


I am having some trouble sorting HUH-7 human liver cells.  The post sort
viability is quite low, and I have to interrupt the sort every hour or so to
remove the filter tips I am using because they are plugged up with cell
clumps.  I make the sample filters by welding 53 um nylon mesh to the large
end of a 20 uL pipette tip.  (I'm using a 100 um nozzle.)  My collaborator
needs to collect as many cells as possible each day we do this.  The
population we are looking for ranges from 0.5% to 5%.  The cells are
suspended at 1.5 to 2e6/ml in dPBS without Ca++ or Mg++.  The sample tubes
are kept on ice before they are mounted on the sorter and they are jacketed
(4 C) while sorting.  I am running a Vantage SE at 9 psi,  21 kHz, Normal-R
with a 2 drop envelope.  The cells are being sorted into 15 mL conicals with
10 mL of F7 medium supplemented with FBS.  I do not have the collection
tubes water jacketed.

Any suggestions for decreasing clumping and/or improving post-sort viability
would be greatly appreciated.


Mark A. Miller
Pharmacology Cell Sorting
Lab WP27E-120
Mailbox WP26-265
Phone (215) 652 4597




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