I am currentlyisolating human monocytes on (Ficoll method) and then purifying by means of negative selection with a stnadard micro bead kit. I want to do co culture with fibroblasts, but I am in doubt about how low in density I can culture the monocytes (monocytes being stimulated with mCSF).
We usually seed 100.000 monocytes per well in a 96-well plate for either direct stimulation or differentiation purposes. If you go much lower your cells will probably die since they need cell-cell contact.
By the way, cheap alternatives to negative bead selection are doing adherance of you PBMCs after isolation followed by washing with PBS (about 70-80% purity). Or doing a percoll isolation of the PBMC fraction after the ficoll isolation (>90% purity).
I support the previous answer. My experience, doing percoll after ficoll worked nicely.
Gentlemen! Thank you for your good advice. Im very interested in the Percoll isolation after Ficoll isolation - would you have a specific protocol, or is it a standard procedure?
I think the protocol is general, depends on what kind of cell type you want to isolate, and to make sure where is it in the system. Just make sure that you do the centrifugation with brake off. Attached please find a nice protocol as the reference I used.
One thing about the percoll though; in the Netherlands you need to sign a disclaimer that you're not going to use percoll to perform IVF (apparently it has a use there as well) I don't know if this is specifically due to Dutch laws, but at least here ordering can take a couple of weeks...
We usually seed 1.000.000 monocytes per well, in a 6-well plate, and stimulate them with 100ng/ml MCSF.
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