Thioglycollate elicited macrophages

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StrangeKitty
StrangeKitty's picture
Thioglycollate elicited macrophages

Hi,
i am quite new to this topic and i could really need some help.
What i want to do is inject thioglycolate (i got the brewer's medium) i.p and extract macrophages 3-4 days later to isolate RNA for receptor studies. So here are my questions:
1.How much TG will i have to inject? (I have read about ranges from 0.1 to 3ml)
2.How do i extract the macrophages? When we tried it once in our lab we injected ice cold cell culture medium into the peritoneum but we couldnt get it back out, because the bowels were always aspirated.

I would be very thankful if anyone could help me out with a protocol!

Thanks!

heehawmcduff
heehawmcduff's picture
Hi and welcome to the forum.

Hi and welcome to the forum.

This subject has already been discussed and there are some valuable pieces of advice in the following postings:
 
http://www.scientistsolutions.com/t53-peritoneal+macrophage+cell+preparation.html

http://www.scientistsolutions.com/t11314-what+is+the+right+thioglycolate+to+use+to+harvest+mouse+macrophages_.html

Regarding your questions, I've seen a few papers where a single injection of 4% TG solution was used and 1ml of the solution was injected i.p. (depending on mouse strain difference, you may have to use a little more).

For extraction of the macrophages, it's difficult to describe without showing you, but there are some detailed descriptions of the technique in the post links above.  The only piece of additional advice I can offer is, make sure you use a wide bore needle for collection of the cells and don't scrimp on the volume of culture media you inject.