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Alice Longobardi Givan, Flow Cytometry: First Principles, pp. 34-40.
This is an introductory overview of the concepts used in sorting.
Howard M. Shapiro, Practical Flow Cytometry, 2nd ed., pp. 106-114.
More detailed approach. A newer edition is available, but I don't have it.
Tore Lindmo et. al., Flow Sorters for Biological Cells, in: Flow Cytometry
and Sorting, 2nd ed., Melamed et. al. pub., pp. 145-169.
Probably the most complete reference for sorting.
All of the above references include references. (Sorry about the 'incomplete'
references; if you have problems finding these, I can give you complete,
Cytometry-format references).
We also have a FACS Vantage. The papers that were published that included
data based on sorted cells from our lab usually address the purity obtained.
If I remember correctly, papers based on cells that were sorted with our
previous cytometer, an EPICS 753, referred to "95% or better" purity. Some
papers based on cells sorted with the Vantage refer to "98% or better" purity.
(If the Vantage is set up correctly, I usually get close to 99%, but if one
sort out of 10 comes back with 98%, then we have to say 98% or better for the
complete set of 10 sorts).
Now, that is sorting under optimal conditions. I also sort cells for a group
from the University of Toledo under non-optimal conditions. First of all, the
distinction between negatively-stained (right sort) and positively-stained
(left sort) cells is terrible! The positively-stained cells are defined by
the "upper shoulder" of a fluorescence histogram, having fluorescence intensity
greater than the control histogram; i.e., there is no separation between a
negative and a positive peak, as staining produces only one peak. The
negatively-stained cells are defined as cells having fluorescence intensity
less than the mode (peak channel) fluorescence. Since positively-stained cells
rarely have fluorescence this low, the negatively-stained cells are collected
with >98% purity. However, negative cells are unavoidably present in my
left sort window, so I get maybe >50% or >75% purity (I can't remember the
actual figure offhand). I also sort the cells at relatively high speeds,
somewhere between 6000 to 8000 cells/second threshold. The higher sort rates
produce lower purities, but really effect recovery (yield) more than purity.
A big factor effecting purity on the Vantage is the drop delay offset (ddo).
This is set on one of the electronic boards that "live" under the lasers. I
think the manual explains how to perform a drop delay matrix, but doesn't go
into actually setting the ddo. (You don't have to set the ddo, but it makes
life easier than trying to remember that you must set your drop delay 1.25
drops less than whatever number AutoSORT comes up with.) Also, there are two
ddo settings: one for standard, rectangular sort windows; and one for SEM,
non-rectangular sort windows.
Another purity factor is sidestream formation (phase). If your sidestreams are
not stable, i.e. the phase is incorrect, then you will not collect what you
programmed the cytometer to collect. This is especially a problem on long,
several hour sorts, when there is more chance that your breakoff distance
might change (as a result of sheath pressure changes that result from the
consumption of sheath fluid).
Another observation is this: sorting non-fluorescent populations from fluor-
escent populations (a "negative" sort) usually produces better purity. This
is probably due to the fact that postive-negative doublets are detected as
positively-fluorescent (and other reasons?).
/\/\/\_ Eric Van Buren, vanburen%flovax.dnet@rocdec.roc.wayne.edu
\ \ \ Immunology & Microbiology
\_^_/ Wayne State University, Detroit, Michigan
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