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
No need to MACS separate. After Ficoll centrifugation take the entire population (unless you are going to do some type of autologous stim with T cells) and pop them in a flask with your culture conditions. After a week the cells that have adhered to the flask are monocyte derived DCs. The ones that are in suspension are primarily macrophage-like (note that to get monocyte-derived Macs you don't need to add IL-4, just GM). After one week, there may be some un-differentiated monocytes, but that is highly unlikely.
You may be collecting the two different populations (adherent cells and suspended cells) to compare cellular characteristics.
Reverend Doakes is correct that you can analyze the purity of these cells by CD11c and HLA-DR expression, further these cells are activated and express B7 molecules (and CD83).
Good luck!
PS. You will never be able to see the dendrites from a DC under these conditions. Even activated DCs, 'shrivel-up' into spherical cells under these conditions.