Optimal sheath fluid for cell sorting

Joe Trotter trotter at scripps.edu
Fri Aug 7 23:57:07 EST 1998


I've got two comments here:

	1) It is worth mentioning at this point in the thread that
sheath buffer can be an issue. Is was a couple of years ago that
a few lab mangers stated they were "saving money" by using water
(dH2O) as sheath in their FACScan instruments. One shouldn't forget
that refractive index is a major player in optical measurements
on cytometers. With PBS + protein as sample buffer and water as
sheath there is a dynamic lens at the buffer interface such that
scatter and fluorescence measurements are distorted. So, one
should try and "match" sample and sheath buffers in terms of refractive
index to a reasonable degree. For example, several investigators here
measuring yeast DNA in a citrate sample buffer swap sheath tanks
and run citrate sheath buffer to improve CVs. Similarly, we sort
chromosomes in the same KCl buffer the sample is in, not PBS.

	2) It is not uncommon to have pH problems when sorting into
96 well plates seeded with unmodified culture media. Here, we advise
facility users to buffer their media with 25mM HEPES so the pH won't
climb too high in room atmosphere. We also recommend 25mM HEPES in
the sample buffer - PBS is just not enough under some conditions.
The best cultured cell viability seems to occur when users bring their
cells in PBS or Hank's with 25mM HEPES, 0.5 to 2% BSA, and 
1mM EDTA. 
 
     Tip: If there is no HEPES added to the tray media we usually toss
some dry ice pellets into the bottom on the sort chamber below the ACDU to
get CO2 around the 96 well plates during the sort. Some cells are more pH
sensitive than others, but HEPES has "fixed" viability issues in many
sorts here over the years, especially when users want to sort into DMEM or
RPMI culture media.


Joe Trotter 
Director, Flow Cytometry
Mailstop Imm-20
The Scripps Research Institute
10666 North Torrey Pines Rd.
La Jolla, California 92037





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