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Preparation of human monocyte derived dendritic cells by plastic adherence.

dendritic cells MDDC plastic adherence

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#1 Sergey Bond

Sergey Bond

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Posted 28 June 2012 - 09:35 AM

Hi, I would like to obtain a reliable protocol for generation of monocyte derived dendritic cells (MDDCs) by plastic adherence. Here I describe my protocol, what I observe and my thought on what I think is happening on the plastic:

1. I prepare PBMC using centrifugation in Ficoll, count cells bigger than 5,4 um on Coulter counter. Normally I get around 30-100 mln of cells from 40-50 ml of blood.

2. As recommended by people who use DCs for clinical purposes ( Sustained expansion of NKT cells and antigen-specific T cells after injection of -galactosyl-ceramide loaded mature dendritic cells in cancer patients, David H. Chang, et al., note Steinman himself is a co-author, JEM Vol. 201, No. 9, May 2, 2005, 1503–1517) , I use Thurner et al. method for MDDCs generation (Generation of large numbers of fully mature and stable dendritic cells from leukapheresis products for clinical application, Journal of Immunological Methods, 223 (1999), 1–15).

3. I plate PBMC on plastic for one hour, carefully remove the media (no shaking, just slightly tilt the plate and aspirate the media, I do not re-plate non-adherent cells as suggested by Thurner just because I have enough of PBMC to spare them).

Notice: No washing is performed. I tried very gentle wash using PBS without Ca++ and Mg++, washing significantly reduced the number of DCs in the end, no matter how gentle I was. May be I should try using PBS with Ca++ and Mg++? But this was not advised in several papers. Most referenced paper on DC preparation uses "a very gentle wash" (J.Exp.Med., 1994, Vol 180, 83-93, by Romani et al.), but I was not able to be so gentle.

4. I add a fresh media (I use RPMI 1640 with different types of serum).

5. Next day I carefully replace this media with a fresh media containing cytokines (GM-CSF and IL-4). You can add the total recommended amount of cytokines in one addition or, if you want to replace the media every other day, you may divide them in 2-3 additions.

6. On day 4-5 of incubation with cytokines I observe floating immature DCs, and almost all lymphocytes are dead! My guess is that lymphocytes are dying from the absence of IL-2. So I gently take of floating non-adherent DCs, count them and re-plate them on a new plate with a fresh media.

7. From my experience I speculate that it is very important to catch the window (4-6 days) when DCs are floating and lymphocytes are not observed, and re-plate DCs on a new plate, because if I miss the window lymphocytes re-appear as a majority of cells. I can only speculate why it is happening – may be DCs express IL-2 and lymphocytes are proliferating from the remaining live clones.

8. Re-plated DCs can be used directly for your experiments. I nether observed lymphocytes in majority after DCs are re-plated.



Please share your knowledge and experience, or your thoughts on what is happening on the plastic. Do you recommend using a special kind of plastic?





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