Hello Benjamin,

I have generated DC derived from human monocytes in a 6-day protocol too, I apply 2 doses of GM-CSF and IL-4 in alternating day and leave the same medium when I apply my antigen so that the DCs matured. If there are any signs of acidification shown by the pH indicator in the medium, I replace half of the medium with tempered culture medium.

Best regards,

Yareellys Ramos Zayas

Hello, after 6 days your cells should be fully differentiated (check by FACS.. >90% should express your markers of interest cd11b). You have to wash them anyway to replate them for the experiment with the TLR ligand. Don't add gmcfs or other cytokines in the media when you add the TLR ligand (if you add them, then you can't say that the effect that you see is not related to the cytokines). Also your controls should not have gmcfs (untreated cells). If you have spare cells, you could add 2 samples where you add the cytokines (untreated and treated with TLR ligand...just to see what happens). Regards

Hi Benjamin,

We have achieved good results with the following procedure. The monocytes were cultured for 5 days with GM-CSF and IL4 (6-well plates). Then we determined CD1a and CD14 as well as CD86 for our purposes. A new seeding was done in 24 or 96 well plates. The advantage of reseeding is that dead or not so fit cells are removed and the cell number can be readjusted. Attached is a paper from our working group.

Best regards

Jochem

Salut Benjamin,

De mon expérience, oui il faut les laisser. Surtout le gm-csf qui va permettre aux cellules de survivre.

Sinon, je ne sais pas ce que tu vas faire, mais 24h de maturation, c'est généralement suffisant.