--- On Mon, 13 Oct 97 11:11:55 MST "Martin, Jill V." <martin.jill@MAYO.EDU>
wrote:
I have an investigator who wants
>to look at leucocyte markers using unlysed whole blood. He says that was
>the protocol used at the institution where he used to work.
If anyone who reads this knows if
>and how the samples can be stained and analysed I would appreciate hearing
>from them.
Certainly a lysing step is used by the huge majority of
investigators examining blood cells. There are a few ways to differentiate
the white cells from the red cells, however, they will take away a
fluorescence channel:
1) you could label with CD45, then gate on all 45+ events which would
exclude rbc
2) you could label with glycophorin, which labels rbc, then gate them out
3) you could use a nucleic acid binding dye which would not label rbc to
exclude them
4) it may be possible in certain cases to separate them by scatter,
especially if the samples can be run unfixed, of course running live cells
brings up another set of issues [timing, availabiltiy of machine, biohazard,
etc.]
Another drawback to this technique is your file sizes will be enormous due
to the relative ratio of rbc:wbc. If your machine has the capacity to use a
live gate and only save the gated events as a file this would help a lot.
There are many commercial an d homemade lysing solutions available with the
advantage of eliminating any rbc contamination of the data. Maybe a quick
comparison exp. [lysed vs unlysed] would allow you to determine which will
work best for that system.
Good luck,
Tom
--------------------------------------------------------
Thomas W. Mc Closkey, Ph. D.
Director, Flow Cytometry
North Shore University Hospital
Biomedical Research Center
350 Community Drive
Manhasset, Long Island, New York 11030
ph: 516-562-4844 [office]; 516-562-1135/4641 [lab]
10/16/97 12:33:56 PM
E-mail: thomasm@nshs.edu
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