large particle sorting
Dave Coder (dave@nucleus.immunol.washington.edu)
Wed, 21 Jul 93 11:14:41 -0700
We're just starting to do large particle analysis/sorting with a
FACStarPlus equipped with MacroSort and the 400um diameter orifice.
After fitting the 400um nozzle, lowering sheath pressure to about 2
psi, and realigning the instrument using chicken RBCs, we get the
expected scatter (FSC and SSC) and fluorescence distributions
(albeit, less precise). At a pressure of 2 psi, the measured stream
velocity is about 2 m/s, which correlates well with the expected FSC
pulse width.
Corn pollen (from PolySciences), about 90um in diameter, was used as
a large test particle. The roughly 60us pulse width for pollen was
as expected (beam height is assumed to be 20um at focus). However,
there were substantial problems with clogging, hence, a sample tubing
with larger internal diameter (and about the same outside diameter)
is needed. Any reliable sources?
Further, sample settling is, of course, a problem and the informal
documentation recommends against using sample agitate while sorting,
but suggests the addition xanthan gum at 0.1% concentration. Any
experience with this vis a vis, fluidics effects (viscosity of the
sample stream will go up), sorting efficacy, sterility of sorted
cells?
Any other gems of wisdom in sorting large particles on the FACStar?
Dave Coder
Cell Analysis Facility
Univ. Washington
Internet: dcoder@u.washington.edu
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