Isolation of monocytes

Marion Freistadt mfreis at lsumc.edu
Tue Jun 28 17:19:52 EST 1994


>Netters,
>I plan to isolate monocytes from a large amount (@ 2 l) of porcine blood. I
>would like to know if anyone is doing work of this nature  and what method
>would
>be the most efficient. Also, what medium is required for culturing these cels?
>
>Thanks,
>Michael Johnson
>
>johnson+c_michael%a1%mallinckrodt at mcimail.com


I don't know about porcine, but for human blood your options are:

1) After ficoll purification, adherence to plastic.  About 70% pure.
2) Flow cytometric with markers.  Very pure, but very slow and low yield
because of size heterogeneity.  Also, the marker may interfere with
function.
3) Flow cytometric by size.  No experience.
4) Magnetic beads.  We are working on this. so far it is not better than
the other methods, but we just started. Contact the manufacturers for their
claims.  Dynal beads, Advanced magnetics or Harlan scientific.  Maybe
others.  You can do negative or positive selection.  Streptavidin-coated
beads and biotinylated primary antibodies may be the way to go, if you have
the porcine markers.
5) Nycodenz.

Primary monocytes will die after about 5 days in culture.  You grow them in
RPMI plus serum (?porcine).  They may do better in Iscoves plus serum or
some of the defined media, especially for monocytes. My understanding and
experience is that anything that likes RPMI likes iscoves better (eg
hybridomas) but I haven't tested it on primary monocytes. They will still
die no matter how happy they are.  If you want immortal monocytes you
either have to transform them or get lines from the ATCC.  I don't know if
they have porcine lines, but you can look it up as per the discussion of
on-line ATCC.

Please forward other responses to the net, because I am interested to know
if there are other people responding to this question.  We've been trying
to get very pure monocyte cultures with varying  success.

good luck.



Marion S. Freistadt
Department of Microbiology, Immunology and Parasitology
Louisiana State University Medical Center
1901 Perdido St.
New Orleans, LA. 70112

504-568-4071




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