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
Phone (215) 652 4597
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