Try Accutase, it works well on our more fussy cell lines and primary cells that are killed by Trypsine.

A cell scraper might also help.

I remember of this paper:

Curr Protoc Immunol. 2008 November ; CHAPTER: Unit–14.1. doi:10.1002/0471142735.im1401s83.

The Isolation and Characterization of Murine Macrophages

Hope it could help!

Accutase I works fine for human THP-1 and primary macrophages so it may be worth to try it for avain macrophages.

http://www.promocell.com/products/reagents-and-supplements/detachment-reagents/accutase-solution/

PromoCell is also offering a special reagent for the detachment of macrophages, but I have no personal experience with this reagent:

http://www.promocell.com/products/reagents-and-supplements/detachment-reagents/macrophage-detachment-solution-dxf/

You should avoid cell scrapers for macrophages which are really sticky cells!

Make sure to use non-tissue culture-treated plates while culturing your cells if you're not already, then the removal should be much easier.

I agree with Jillian, you can use various methods to detach them (trypsin does work as well, I usually scrape) if you use non-tissue culture dishes!

I agree with Victoria on the Trypsin method, namely it will kill the cells before they will detach. Like Monika, I suggest you scrape them (that is what I used to do). You can however also try lidocaine or Accutase I. Still if you need the cells for flow cytometry I definitely would not use any enzyme, but simply just scrape them.

Dear colleagues, in my experience scraping and lidocaine cause a large number of dead cells; you might try Upcell plates from Thermo. These plates are expensive but work quite nice for gentle detaching cells. Best regards Stefan

I've recovered 90% of my adherent macrophages (plated for 6-10 days, so extremely adherent) with TrypLE Express. It is an extremely gentle, recombinant protease that does not cleave surface antigens - which is important to me as I needed to phenotype my cells by flow cytometry. In my experience, TrypLE works much better than accutase and scraping as the cells are still viable and lift quickly. Here's a paper comparing differing reagents to lift adherent endothelial cells, and I had the exact same findings for macrophages.

http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.22683/abstract?systemMessage=Wiley+Online+Library+will+be+unavailable+on+Saturday+27th+February+from+09%3A00-14%3A00+GMT+%2F+04%3A00-09%3A00+EST+%2F+17%3A00-22%3A00+SGT+for+essential+maintenance.++Apologies+for+the+inconvenience.&userIsAuthenticated=false&deniedAccessCustomisedMessage=

Good luck!

Lifting adherent macrophages is pretty difficult, especially primary macrophages, although cell lines like THP-1 and raw 264.7 cells are a lot easier to work with. I have tried a lot of different methods for lifting primary human macrophages and nothing works particularly well. Which method you use really depends on what you are trying to do afterwards. If you want to analyze them by FACS and are going to fix them, you need to make sure the surface antigens are intact but not necessarily , whereas if you want to replace them and use them for subsequent assays you need to keeps the cells relatively happy so they will respond normally.

You are right that trypsin is very hard on macrophages, and it also strips or damages a lot of the surface markers so I would not recommend it.

When I was a grad student, I would put the cells at 4°C for about 30 minutes, then add cold Versene (a 1:5000 dilution of EDTA) and hit the flask with my hand. This dislodges most of the cells but is a bit rough - you recover most of . Similarly, putting the cells at 4°C and pipetting up and down with cold PBS, followed by gentle scraping with a cell scraper also dislodges the cells. Both of these methods will leave the cells a bit "unhappy" and you will get some cell death - maybe 25 - 40% - but if you replace the remaining cells and leave them overnight they perform well in a variety of assays.

I have not had much luck with accutase - I find it does not lift primary cells well at all - and agree with the earlier poster that TrypLE / TrypLE express works much better - although I have found that you still need to pipette up and down a lot to get your cells off, and that you get between 50 - 75% recovery.

If you are interested in changing the culture plates you use, Upcell plates do work pretty well - although I have found that assays I perform on Upcell plates do not always act the same way as on standard tissue culture plates. I would not recommend using non-tissue culture treated plates as macrophages plated on plastic are activated and mature in a certain way and I am not sure the same process occurs without the tissue culture treatment. You could also try plating the cells on glass, to which macrophages are much less adherent, but again, this might interfere with the maturation process you are used to seeing.

Overall I use TrypLE + pipetting and sometimes scraping when I need to analyze my cells by FACS, and simply plate and mature the macrophages in the plates I will ultimately perform the assays in when I need to perform functional assays. This can be problematic because plating down the same number between assays is difficult, but can be somewhat mitigated by staining for CD14+ in the PBMC population before plating and normalizing that way, or if using the cells for infection, performing the infections with an MOI based on the CD14+ percentage in the PBMC.

Perhaps you should try Accumax (similar to Accutase but more concentrated). 

I'm not sure about primary macrophages, but we routinely grow macrophage cells lines such as RAW in bacteriological dishes. They don't adhere so strongly but are still perfectly happy and proliferate well. You can dislodge them with a stream of medium or a gentle tap.