I culture my human monocyte derived macrophages in Nunc Upcell® plastic plates and lift them in 5mM EDTA/PBS at 4 degrees, and am able to lift most of the cells successfully. However, because of the assays I do, they need to go back into a 37 degree atmosphere which gives me the idea that they are clumping (noticed when counting the cells in a haemocytometer)
resuspend them is indicated in the first answer in a medium condaining EDTA and if you want to inject them into mice you can use a droplet of Heparin
Would a little heparine be a good idea? I used to add heparine to retrieve macrophages from the peritoneal cavities of mice. Without heparine, they would form clumps indeed.
I'm doing an in vitro assay so I'm not injecting into mice but the heparin sounds like a good idea. would have to read about any potential effects on macrophages
Hi Shaumick!
Do you really need to lift up your macrophages and then plate them again? Can't you differentiate them already in the final plate where you'll do the assay? I work with murine bone marrow-derived macrophages and I do the differentiation protocol in the same plate where I perform my infection assays, so I won't lose cells nor activate them. My macrophages usually don't proliferate, just differentiate from progenitors, so I assume that the final number is the same as in the begining. Anyway, if you really need to lift up your macrophages, EDTA in cold culture medium without FBS usually works fine, giving good viability and not so much activation. But if you wish to do any surface staining watch out, because you may lose some markers. Maybe if you wash your cells immediatelly and resuspend by pipeting up and down against the tube wall your macrophages will clump less.
Hi Joana,
Sadly I do need to lift them. Usually, after checking on flow, the markers I look at are preserved. One thing I did realise after reading about is that actually the clumping could be from cell death which seems possible because a colleague did confirm that these cells are very sensitive once lifted
But when you count the cells on the haemocytometer, I supose you use trypan blue or other vital staining. The cells you see clumping are the death cells? Or refringent live cells that aggregated?
I counted again (this time with trypan blue) and yeah dead cells
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