Hello everyone,
I´m isolating monocytes of peripheral blood to differentiate them to macrophages. I am isolating the PBMCs by Ficoll centrifugation and after that I plate them on a 10 cm petri dish. Usually I plate 60 x 10^6 PBMCs in 10 ml RPMI with 10 % FBS and 1 % P/S. I´d like to separate the monocytes from lymphocytes and other contaminations by plastic adherence of the monocytes. But the problem is that the cells don´t adhere probably on the plate. So I can´t get rid of the lymphocytes only by medium change. Does someone know the reason why monocytes don´t adhere or has someone any recommendation for monocyte isolation by adherence?
And besides that, I´d like to know which macrophage yield to expect when separating monocytes by plastic adhesion and cultivation for 7 days?
Thank you!
I also make MDMs. I usually seed PBMCs in complete media, for overnight. Next morning, I use to remove all non-adherent cells and wash with DPBS.
It remove almost all lymphs.
If you are using serum free media the 6 hr is good time for adherence.
Hi Johanna! I noticed that you plate you cells at quite high concentration. I also use this protocol to enrich for lymphocytes, but I usually seed PBMC at 1×10^6/ml in big flasks and I usually observe a good adherence. Possibly, the high cell concentration interferes with adherence
Are you using the tissue culture treated plates?
If you are using non treated plates then monocytes or any cell type will not attach to the surface.
Thank you all for your advice!
Sudeep Kumar I am usually using non treated plates. Yesterday I did another run and seeded PBMCs into a non treated plate. Cells did adhere perfectly after 2 h, but cytospin showed that 97 % of those attached cells had been lymphocytes instead of monocytes which I really don´t understand. By using treated culture plates, lymphocytes would adhere even more strongly, wouldn´t they?
Nicole Acuff I let them adhere for 2 h in serum-free RPMI medium
Hi again Johanna! I read your answers and I noticed my protocol is quite different. We use treated flask and let monocytes adhere overnight. If they're not activated, lymphocytes usually don't adhere, and a couple of gentle washes with PBS is sufficient to remove them. I hope this can help you!
Another thing, we add 10% FBS to medium. For my knowledge, it is mandatory for attachment
I agree with Costanza Maria Cristiani , you will need both a cell culture treated plate and 10% FBS.
If at all lymphocytes adhere, their adherence is loose and can be dislodged by a couple of washes with medium.
As I read, non-treated plastics doesn`t support cell adhesion. Once by mistake I seeded cells into a non-treated flask and they didn`t even attach. So try tissue culture treated plastics and better with high serum
I use polystyrene plates, for cell culture (48 wells). For cell adhesion, the cells are incubated for 1 hour and the plate was shaken at 30 minutes’ intervals to avoid non-adherent cells (Lymphocytes) precipitation. The supernatant containing the non-adherent cells are removed and deposited in polypropylene conical bottom tubes (I use, tubes BD Falcon).
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