[Cytometry] Advia or Isoton
hms at shapirolab.com
Sun Feb 28 14:33:36 EST 2010
Akos Szilvasi wrote:
> Almost five years ago we switched to water as sheath fluid on our three
> LSR II+HTS cytometers. We have not seen any negative effect except once
> one of the instruments started cumulating air bubbles in the nozzle. After
> extensive trouble shooting with BD the solution was to add a little
> surfactant (detergent) to the tanks. Our tanks are the 25 liters steel
> cylinders from BD. Now we regularly add about a ml of FacsRinse to a tank
> of distilled water (never use tap water).
> The advantage of this practice is the we practically eliminated all salt
> deposits related HTS (BD's great 98/384 well plate samplers) problems, do
> not buy and store PBS or sheath fluid (except, of course, for the sorters)
> and the filling of the tanks is a simple task.
> The critics of this practice mention the difference is refractive index
> and increased low scatter noise. The water goes through the LSRII's 0.2
> micron filter and in my experience both potential problems are negligible
> (if measurable at all).
My colleagues and I have been using distilled water as sheath fluid for
our analytical flow cytometry since the 1970s; we now generally run
"bought" distilled water through our MilliQ system, which includes a 0.2
um filter, before putting it into our sheath tanks, and also keep such a
filter in the sheath line. Even when we are measuring physiological
parameters, e.g., membrane potential, the interval between injection of
the core sample into the sheath and the interrogation of the sample is
short enough that we do not see any changes attributable to the
osmolality mismatch, and scatter noise is only an issue when we deal
with particles smaller than most bacteria, if then.
I would guess that one could get away with using lower than customary
salt concentrations in sheath (just enough to maintain conductivity)
even when sorting, since the small amount of hypotonic solution in a
sorted droplet will quickly be diluted by the more nearly isotonic
medium into which cells are collected, but I will defer to those with
more sorting experience on that issue.
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