Isolation of monocytes

[Marion Freistadt]

>>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