What is the best way to generate iDC from monocytes? There are many easy protocols for these, but they do not describe the plates e. g. adherent, nonadherent?
They usually are in suspension after 5-6 days of culture. I use for these exps 6 or 24 well plates, 1x10^6 cell/ml.
Excellent question! We've had very poor results with tissue culture treated plates. Instead, we use 10cm polystyrene petri dishes (Fisherbrand 0875712). Good luck!
We have no problems with using T25 and T75 polystyrene tissue cultre flasks from Sarstedt for human DC cultures. 6-48 well polystyrene plates can also be used; but not so much with 96 well plates.
You can harvest the non-adherent DCs by removing the cell culture supernatant from your 5-6 days DC culture. To remove adherent cells, incubate them with EDTA-saline solution with 1mM glucose for up to 10 minutes at 37oC. Hope that helps!
Hi... we are using T25 Tissue culture treated flask. and getting good yield. But you should keep remember your rm-GM-CSF dose.
Are you trying to generate DC1 for immunotherapy or DC2 for regulatory responses? The culture surface will direct that. For TH1, T25 or T75 polystyrene is best. Some say laminin, not matrigel, will give better IL-12p70 output. For DC2, you can culture in bags as the polyethylene is taken up by the DC and locks out TH1 gene expression. Easier to harvest, too, as no adherence problems.
Good Luck
For DC in culture, probably a maximum of 7 days before they die off. If you want to freeze them, try 10% DMSO with Human Serum Albumin at 2%. Controlled-rate freeze to -80 C, then after overnight move them to vapor phase of LN2
If you want to culture DCs in glass-coverslips, you can pre-coat the slips with 2% geltin for 2-3h before you add cells.
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