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What should be and what is are frequently not the same. As one person
put it "theory collides with reality and does not fair well". Having
tried this on a number of occasions, it's clear that "going faster" can
definately decrease sort purity. The problem is with the speed of the
electronic signal processing. The electronics on most cell sorters are
incapable of detecting all the cells flowing through - so even with
coincidence checking you can get impurities at even moderate rates of
say, 3 to 4,000 cells/sec. The machine can't exclude a cell if it
doesn't see it.
With beads I can acheive 4-nines (1 or less beads in 10,000) if the
populations are approx. equal in percentage and I keep the trigger rate
under 1000/sec.and spend 15-20 minutes cleaning the sample tubing before
reanalyusis.
If I try sorting out a 1% population at rates exceeding 4,000/sec., I'm
lucky to get 96-98%.
>I therefore strongly endorse the suggestion of re-analysing all sorted
>fractions.
Agreed, whenever sufficient numbers of cells are available.
tom d.
David Abbott's thought for the day:
"Many people stop looking for work when they find a job."
-- ============================================================================== Thomas Delohery | Internet: t-delohery@ski.mskcc.org Manager, Flow Cytometry Core Facility | Phone: (212) 639-8729 Memorial Sloan-Kettering Cancer Center | Fax: (212) 794-4019 ==============================================================================
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