RE: rbcs and dead cells in post sort samples

Hi Jacob,

 

It may not be an issue of the cells sticking to other cells, but rather
an issue of dead cells and red cells falling below your FCS threshold
which means that the sorter is basically blind to their presence. Upon
reanalysis, they happen to fall above the threshold and you detect them
as negative events.

 

While your users worry about decreasing their yield due to extra
manipulation, they are probably seriously decreasing their yield due to
the presence of these negative events. These negative events (those that
fall above the threshold on the sorting pass through the XDP) will cause
an increase in the number of software aborts which will decrease the
number of desired events that are sorted. The more unnecessary negative
events then the greater this effect. Decreasing the threshold level to
visualize these cells and then gating them out will not minimize this
software abort effect, in fact it will increase it as you are detecting
even more unwanted events in close proximity to your desired cells in
your droplet queue which leads to software aborts.

 

The XDP has the ability to collect the software aborts into a separate
tube while sorting in purity mode, so you could try collecting the
software aborts and resorting them after the first sort. The abort
collection tube should have about 60% purity after the first sort which
will minimize the software abort problem on the second pass through the
sorter.

 

Karen Helm

University of Colorado Cancer Center

Flow Cytometry Core

 

 

________________________________

From: Blair, Jacob Roberts [mailto:BlairJR@health.missouri.edu] 
Sent: Monday, June 23, 2008 4:26 PM
To: cyto-inbox
Subject: rbcs and dead cells in post sort samples

 

Hi everyone,

 

We have a few customers who bring samples that contain a lot of dead
cells and red blood cells.  The customers prefer not to lyse the rbcs or
put their cells through a gradient to remove the dead cells and debris.
They worry about decreasing the yield with the extra manipulation.  We
think that the rbcs and dead cells are sticking to our live cells and
are sorted along with the population (giving us a lower purity).
Depending on the downstream application, the contaminating rbcs/dead
cells may negatively impact the results.  Does anyone have suggestions
of ways to increase the purity without changes in sample prep?	We are
having a difficult time excluding them with any kind of doublet gating
using a MoFlo XDP.

 

Any suggestions would be greatly appreciated!

 

Thanks,

 

Jacob R. Blair

Research Specialist 

Cell & Immunobiology Core

University of Missouri, Columbia

One Hospital Drive, M324 MSB

Columbia, MO 65212

Phone: (573) 884-7315

Fax: (573) 884-0665

Email: blairjr@health.missouri.edu <mailto:blairjr@health.missouri.edu> 

Web: www.biotech.missouri.edu/cic <http://www.biotech.missouri.edu/cic>