Dendritic cell isolation - (Jun/05/2002 )

I am working on the dendritic cells isolation from the PBMC. The method that use in our laboratory to let the monocytes to attach to the plastic culture flask for one hour and then wash away the non-adherent cell. After that then further incubate at 37 degree to for overnight. The cells would be harvested and culture in IL-4 and GM-CSF for the dendritic lineaged out.

But the problem is I always find a lot of lymphocytes still attach at the bottom of the flask even after the wash step. Moreover, after the overnight incubation I find a lot of monocytes that still attach at the bottom of the flask that cannot be washed out. These two problems both reduce the quality and quatity of the DC isolation. Are there any method to solve them, please?

I have also noticed that a lot of monocytes and lymphocytes are left in my dendritic cell culture.  The solution appears to be to harvest only the loosely adherent cells after your 5-7 day culture with cytokine pressure.  The remaining adherent cells are almost impossible to remove and show macrophage markers (CD14).  I also have noticed by FACs that this newly described technique in Immunological Methods give you a relatively heterogenous population of cells (at least two if not three cell types based on FSC/SSC scatter).  Although more tedious, an initial isolation of CD34+ cells gives you a better prep of DC.  But I would only recommend this for confirmation after you have made an initial observation.
Please check out my forum thread at "Proteins Without Antibodies"
Thanks...

Is it really no any other method to improve the cell-adherent method because we cannot afford to use the antibody to purify the CD34+ cell first

You could try to isolate the DC population at the end of the culture by fluorescently sorting on a DC specific marker such as DC-LAMP(intracellular) or CD83(extracellular on mature DC).  But this would require access to a sorter.

In order to have a nice Dc prep I recomend the following recipe:
Add 200-250 x 106 cells per flask (T125) in 50 ml of RPMI 5%AB huamn serum. Allow the cells to be atached for 2 hours. Remove the non-adherent cells and wash the adherent cells at least three times with PBS in order to eliminate the small cells. You must look under the scope to visaulize your preparation. Add 50 ml of AIMV suplemented with 10% AB Human serum with GM/IL-4 for seven days. If you want to push the differentiation add more citokine at day 5. At day seven just harvest the non-adherent cells. Wash the flask at least once with PBS. I recommend to nalyse by FACS the prep. You will get >95% DR+ and less than 3% lineage. This type of DC works well for antigen presentation and activation as well. I hppe that you will get nice Dc's.
Alberto
ajrivas@yahoo.com