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We have a method for culturing and differentiating normal human monocytes
that was originally obtained from a friend who was doing similar things with
mouse monocytes. The trick is to culture them in bacteriological petri
dishes---more specifically those from Sterillin. Other dishes do not work
as well! You can remove them by replacing medium with cold PBS w/o Ca++ or
MG++ and letting the dishes sit in contact with a cold surface(on a glass
or metal surface works best ) in a refrigerator. till you see them start to
round up and lift off. We help them along by very gentle scraping when we
harvest. You will still run into problems with the cells sticking
particularly with short term (2-24hr) cultures. In our hands, in vitro
human monocytes/macrophages go through some cycles of adherence and
attachment during their differentiation ( normally we culture them 7-9 days
before removing the macrophages for experiments). It helps to let the cells
sit in complete medium on ice after harvesting to allow time for membrane
repair, before doing a viability check and trying to label for surface
antigens.
Also, not knowing the experimental protocol being used, things like
components of the culture medium and concentrations of TNF being used could
also be affecting viability. This does not address the question of how to
effectively block nonspecific binding to Fc receptors, when you try to label
for FCM analysis.
Good luck, they're not easy cells to work with!
Kathie Woods-Cook
Ciba Geigy, Basle
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From: O=internet; DDA.TYPE=RFC-822;
DDA.VALUE=owner-cyto-sendout(a)flowcyt.cyto.purdue.edu
To: Cytometry Mailing List; woodska1
Subject: Staining of peritoneal macrophages
Date: Thursday, 27. June 1996 22,55
<<File Attachment: BDY10.TXT>>
DATE: Jun 26 15:40:31 1996 -08:00 relative to GMT
IPMessageID: v02110103adf7798459ec(a)128.120.30.153
FROM: (Carol Oxford) [O=internet; DDA.TYPE=RFC-822;
DDA.VALUE=cloxford(a)UCDAV
IS.EDU]
TO: Cytometry Mailing List [O=internet; DDA.TYPE=RFC-822;
DDA.VALUE=cytometry(
a)flowcyt.cyto.purdue.edu]
SUBJECT: Staining of peritoneal macrophages
IMPORTANCE: normal
AUTO FORWARDED: FALSE
PRIORITY:
ATTACHMENTS: c:\tmp\BDY10.TXT
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Hi all,
I have an investigator who has been obtaining peritoneal macrophages from
mice by lavage, culturing them overnight with TNF, and then looking by flow
for Ia expression. We weren't getting much expression so I had him stain
with PI at the same time. It turns out that the vast majority of the
cells are dead. He has tried culturing in suspension or letting the cells
adhere and using cold to detach them. Does anyone have a method of
detaching that is easier on the cells and still leaves surface markers
intact? Any advice is greatly appreciated.
Carol
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Carol Oxford _| _| _|_|_|_| _|_|_|
Medical Pathology Flow Cytometry Lab _| _| _| _| _|
University of California _| _| _| _| _|
Davis, California 95616 _| _| _| _| _|
(916) 752-7205 _| _| _| _| _|
(916) 752-4548 fax _|_|_|_| _|_|_|_| _|_|_|
cloxford@ucdavis.edu
University of California, Davis
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