There is nothing difficult. Usually, you sac a mice, take the spleen (I guess) or the tissue that you would like to harvest cells from, and isolate the bulk lymphocytes from the tissue. You can sort the cells using a magnetic kit or flow. The number of macrophages you want to transfer depends on your purposes and I would do a dosing study to optimize the conditions first, usually around 5*10^5 to 2*10^6 cells per recipient.

No, there is not any established protocol.

The number of cells you want to transfer depends upon your experiment. questions to think about ? How much cells do they have in naive mice ? Are you adding a tons of cells that would be very unnatural ? Are you adding very less so that you won't see the effect. have you depleted the macrophages in the recipient mice ?

Media of cells when you are transferring the mice is also something to be considered.

Attached is an article where I transferred macs to the peritoneum of recipient mice to follow there activity. Good Luck, Charlie

Depending on the source of the macrophages you will transfer, their purity may be an issue, e.g. resident peritoneal cells are ~70-80% macrophages with other cells making up the rest (B1 cells, etc), so if you transferred resident peritoneal cells, you might eventually have to purify your putative cell causing any effect seen. If the macrophages are pure to begin with, this is not an issue.

Thank you very much for all the answers!

To keep you updated:

Thioglycollate elicited macrophages were enriched after 1h in vitro adhesion step in fully complemented RPMI. Then, I stimulated the macs for 24h in vitro, harvested with cell scraper and injected 2.5^6 cells in 200µl PBS per mouse, intraperitoneally. After 12h I performed the actual experiment on the recipient mice.

It turned out to make a difference! :-)

Additionally, eGFP macs without stimulation step could be found in recipient mice after ~20h only in peritoneum, not in spleen, liver or blood.

I will now test that at a larger scale, more mice...

Further Question:

Can the innate "memory" be transferred between individuals via macrophages?

Is that novel or well known? good reference?

I will report on the follow-up experiments outcome.

thanks

Fabian