how to distinguish monocyte and dendritic cell under microscope - (Jul/31/2008 )
i am requested to culture monocyte derived dendritic cells, after obtaining PBMC through ficoll, monocyte is isolated by both MACS or plastic adherence, then monocyte is culture in RPMI with 10% AB serum plus GMCSF and IL4 so as to obtain immature dendritic cell, However, when i look under microscope, all the cells are round in shape, some are stick to the bottom of the dish, some are just floating in the medium, some are clump together, so,, which of them are those immature denritic cell i needed and which of them are monocyte that has not derived into dendritic cell?
i think monocyte should be sick to the bottom of the dish, so we need to collect those cell at the bottom, and then induce it to become immature dendritic cell, but i was told to collect those cell floating in the medium to be induced, why?
i think monocyte should be sick to the bottom of the dish, so we need to collect those cell at the bottom, and then induce it to become immature dendritic cell, but i was told to collect those cell floating in the medium to be induced, why?
After PBMC isolation, collect the CD14+ population (use StemSep or Miltenyi) and culture these cells with GM-csf/IL-4 for 7 days. After this period, do a light wash and collect only the cells that have stuck. these are the DC's. anything else is either not committed to DC or some other random cell you don't want...but probably a monocyte. you can check purity by flow staining with CD14 and B7 and CD11c and class II staining to look at activation of the cells. good luck