To Lish about Macrophages
yager at WADSWORTH.ORG
Fri Jun 5 16:32:14 EST 1998
Sorry it took me so long to reply. I tried to email you back, but I kept
getting returned mail errors. Hopefully, you'll see this posted on the
Bionet site. Anyway, here's the answer to your question: There are a few
ways to isolate murine macrophages. The best way to recover murine
macrophages is to flush the peritoneal cavity with cold sterile PBS. To do
this, carefully disect the mouse as to not tear the peritoneal cavity.
Then, inject in about 5-6ml of cold PBS with a 6cc syringe and a 27G
needle. After that, I gently tap the sides of the cutting board to loosen
any cells that are still attached to the peritoneal wall. Using the same
needle and syringe, insert needle bevelled down and withdraw the peritoneal
fluid. I typically get between 1 and 5 million cells by this method.
Peritoneal macs are mature, resident (and perhaps slightly
activated) macrophages. If you want immature, inflammatory macrophages you
must treat the mouse with an inflammmatory agent like proteose peptone 3
days or thioglycolate 5-7 days prior to harvest.
If you want a more homogeneous population of murine macrophages, I
suggest using bone-marrow derived macrophages. To do this, first harvest
leg bones (femur or tibia both work good) from euthanized mice. Then, flush
out the bone cavity into a tube using a 6cc syringe (27G needle) filled
with either sterile PBS or culture medium. Centrifuge cells down, resuspend
in 3-5 ml of medium, and separate out the mononuclear cells you want by
using a lymphocyte separation media like Histopaque 1077 (Sigma). Remove
the cells from the interface with a Pastuer pipet and poll by centrifuging.
Resuspend the cells in 10 ml of media and aliquot into a T25 overnight at
37C, 5% CO2. After 24h, transfer nonadherent cells to a T75 flask with 10ml
of media supplemented with 20% L929 supernatant (the cell line produces
GM-CSF) or IL-3 (which I don't use). Incubate for 4 more days, add another
10ml of supplemented media, and incubate for another 3 days. I typically
get around 10 million. To detach the cells from the T75, one must use the
protease dispase (CalBiochem) and a scraper.
Both of these techniques were taken from "Current Protocols in
Immunology" Chapter 14 which may be available in a scientific library if
you have access to one. If not, I could fax you the necessary pages. They
describe the procedures in greater detail than I have in this message. Good
luck and if you have any other questions, feel free to email me.
c/o Dr. Gary Winslow
Wadsworth Center for Laboratories and Research
Axelrod Institute Rm. 5083
120 New Scotland Avenue
Albany, NY 12208
(518) 486-4393 (Ph)
(518) 474-2696 (Fax)
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