Murine intraperitoneal macrophages - (Mar/23/2006 )
I'm very unexperienced, so any help would be much appriciated!
We're using PBS to isolate the cells but we get to few cells. The cells are not deviding in culture, is that right? So when I aspirate the media only a few cells remain attached. Next time I will check the viability of the cells in the medium and if they are alive I will seed them again. But still there are only a few cells attaching to each plate. Any tips and experiences anyone? Please???
Thanks a lot!
You should be able to get 3 million cells from the cavity (70-80% macrophages, 20-30% lymphocytes). We lavage the cavity with 2 or 10ml PBS using a venflon and someone in our group does culture the cells for at least a week in supplemented RPMI (plus L-glu and pen/strep) plus 5% FCS. (I'm only starting to do this now).
All the best,
ok, thanks a lot. We get that many cells. The problem is it's not enough for Western blots... I got only about 7 µg of protein from one dish last time. But thanks anyway. Now I know at least that the isolation step works as well as i should.
hi moe ! isolation of resident peritoneal macrophages is a very easy task. but you have to take care of few things.
1. if you want to increase the count try giving some inert material 4-7 days prior to isolation like thioglycolate or starch usually 1%w/v is used . these are inert particulate material and macrophages respond well to particulate material. they reach the site( peritoneum in this case) to engulf the matter. these materials being inert does not ilicit any major responses.these macrophages are calledinflammatory macrophages or ellicited macrophages but not activated macrophages.
2. try using heparin @ 10U/ml in isolation solution. heparin prevents aggregation of erythrocytes and there by loss of macrophages which usually gets trapped in the aggregates.
3. use chilled PBS or incomplete RPMI/DMEM with heparin. cold temperatures prevent aggregation of macrophages and also adherence to centrifuge tubes
you can culture these isolated macrophages for upto a week in complete RPMI 1640 or complete DMEM medium. these cells are terminally differentiated cells and they donot divide.( cos they are not cancerous in nature like cell lines.
for further information you can refer " methods in enzymology " vol. 108 pg 274
i hope this will be of some use for you
all the best