Macrophage culture - (Sep/20/2007 )

Hi,all.

I'm pretty new to macrophage work.
I've got all the protocols for harvesting them, but I have questions about maintaining them.
Macropahges are the ones attached to the dish, right?
Then, how do you retrieve them? Use trysin/EDTA as for adherent cells? or just vigorous pipetting?
For phagocytosis assay, is it better seed them on round bottom or flat bottom plates?

Your help will be just great.
Thanks.

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Yes

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Hi macrophage are not only the adherent cell. You can have some cell line of macrophage who are adherent or in suspension.
For adherent macrophage you detach cell with PBS-EDTA or by scraping and probably trypsin I suppose. It depend of which cell line you use. For example with Mf4/4 I need to use PBS-EDTA to detach those cell but for J774 I need to scrap the dish.
For suspension macrophage just recolt you cell and then centrifuge 5' at 1200rpm.

Hope to help

biofred

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Thank you guys.
I assume trypsin is O.K. for primary cells.

Oh, another question.
What's rough sum of total # you get from peritoneum without injecting Thio?
Some say couple ten thousands, others say couple millions.

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hi, for primary cell i use warm PBS containing 10mM Glucose + 3mM EDTA (EDTA is enough to detach the cells, and the glucose protects them).
Re-peritoneal cells without Thio, i'll say between 0.5 and 5 million depending on the mouse strain and your peritoneal lavage ability.

hope this helps, good luck!

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Hi!


I usually get about 2.5mln without Thioglycollate. I also found that the number correlates to background on which the mice are bred (such as C57BL p53 KO versus wt).


Katya

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