Hello
I´m working with Bone Marrow Derived Macrophages and I want to study their behavior in different conditions when I co-incubete them with apoptotic cells.
To differentiate my cells I grow them in complete RPMI with M-CSF 20ng/ml. To collect them, I use a scraper, but this procedure generates a large number of dead cells, which, I suppose, can interfere with my results, even if I wash the cells after they adhered to the plate. When I collect my BMDM with PBS-EDTA I have a very low yield, also, I´m afraid the mechanical stress will affect them because they are difficult to harvest
So, I wonder if it is posible to differentiate them in a 96 wells plate and use them directly in the same plate.
Have someone already done it?? Which complications could I have?
Thanks
All macrophages, including BMDM, are highly adherent, so to unstick them you must use plates for bacterial cultures or suspension plates. I recommend using Accutase solution or PBS with 10mM EDTA to remove them. Do not use scrapers because you will kill macrophages
And in addition, I think it is possible to differentiate them on a 96-well plate, you just need to reduce the scale of the experiment accordingly
I culture BMDM in 24 plates, 2 x 10^6 in every well, RPMI 1640 with 10% FBS, M-CSF 20 ng/ml. change half media in day 3 and 5, after 7 days i add my stimulant for 24h or 48h, then to harvest the cells add 200 ul stempro accutase cell dissociation reagent for 5 minutes in 37 degree . to stop the enzyme action add 200 ul culture media, if you stimulate M1 it will be more adherent to the plate , so you have to pipette more but not intensely. while M0 and M2 is easier to get.
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