Dear Rodrigo,
Thanks a lot for your valuable answer. Would help me a lot.
A couple of questions:
1. Can we stop at 6 day and use the cells for further characterization as most of reported papers do so.
2. Can you also give me detailed protocol for the PBMC isolation and CD14 selection that you use. Do you use any antibiotics in the medium? I ask this because I got contamination a couple of times and the human samples are difficult to obtain.
Thanks!
By sequential steps
1.Monocytes harvesting by leukapheresis, are differentiated in vitro under GMP-conditions into immature dendritic cells.
-Follow a generalised protocol to separate WBC cells from Whole blood
The usual prototypical cytokines used for the generation of monocyte-derived DCs are GM-CSF and IL-4
-Invitro growth coditions with GM-CSF and IL4 , incubation follows 6 days
-The phenotype resembles Immature Dendritic cell, (No other cell can show this kind of phenotype) like monster moving legs or hands
-Now these cells are Qualified Dendritic cells- By FACS analysis you can differ very clearly, also optimise for secondary evaluation
-Some of the critical markers such as CD 15a, CD15b , will helpful to differ monocyte from dendritic cells.(These two absent in dendritic cells )
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The combination GM-CSF and IL4 generates Dendritic cells in a week of days
Try some papers of Rockefeller university -Noble Laureate- Ralph Steinman
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