sort viability

Gerhard Nebe-von-Caron Gerhard.Nebe-von-Caron at Unilever.com
Wed Aug 26 11:37:07 EST 1998



          Hi James
          
          Whilst the blue light can excite certain intracellular 
          molecules the low energy and exposure time makes it unlikely 
          to take an effect unless the cells are already damaged prior 
          to sorting.  I have done similar experiments in order to 
          test the potential injury inflicted by the laser on 
          heat-injured bacteria that are quite fragile.  I sorted 
          cells either based on the red HENE or the blue Argon laser 
          with similar recovery, thus deciding the wavelength not to 
          be a problem.
          
          Form the literature there is quite some evidence of the 
          effect of cell acceleration on the viability of cells which 
          can be affected by nozzle diameter and differential 
          pressures, but they all seem to be not as dramatic in your 
          case.  However you also might consider flow restrictions in 
          the system that might cause a cell squeeze effect.  You 
          might also want to look at the possibility of sheath that 
          flows back into your sample or potentially bleach from the 
          disinfection run if performed before the sort, so you should 
          consider comparing the viability of the cells in the sample 
          tube compared to the cells in the sorted tube.
          
          I assume you compared the 'unsorted' cell viability also 
          after diluting the cells in your sheath medium at the same 
          temperature as your sort conditions, as that might have a 
          substantial effect on their survival.
          
          Regards
          
          
          Gerhard


______________________________ Reply Separator _________________________________
Subject: sort viability
Author:  cozens at postoffice.utas.edu.au at INTERNET
Date:    24/08/1998 18:36


Dear flowers,
     Thanks for all the helpful responses so far concerning my problem
with obtaining viable sorts. Perhaps this additional information may be of
use.
The cells I am sorting are murine splenic lymphocytes. Directly post sort
their viability is 80% (tyrpan blue).
Flow details: Coulter Elite ESP sorter, power 15mW air cooled argon, 100um
nozzle, sheath straight PBS, sort speed @3000/sec, pressure @11 psi

I've done several test sorts and my results indicate an increase in
viability with the laser off (collecting everything) although viability
still remains low. Is there a possibility that laser power could be
affecting my cells in some way?

The concerned student, James.


Mark A Cozens
Division of Pathology
University of Tasmania
ph     03 62264828
fax     03 62264833



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