And for co-culture studies would CD4+ tcells with our cell of interest be ideal to check for PGE2, IFN gamma and NO expression or PBMC with the cells ?
Human dendritic cells as positive markers for FACS and co culture studies to check immunomodulatory capacity with our cell of interest. The PGE2, IFN - gamma And NO from culture supernatants to be used
cell + PBMC?
or
cell + CD4+T cells
Dear Elizabeth, of course you can use the IL-4 and GMCSF expressed in E.coli. But you need to take care of two things.
1. functionality, i.e. activity of the IL-4 and GMCSF
2. Purity of IL-4 and GMCSF, specially contamination of LPS when you purified from bacterial lysis. LPS can stimulate stimulate wide variety of the cytokines. when you want to measure the cytokines and NO, your IL-4 and GMCSF must be free of LPS.
@vijay thank you, will keep that in mind . Would you happen to know - for coculture studies - CD4 T + or PBMC s would be the better choice ?
We do generate dendritic cells from both PBMCs as well as from bone marrow progenitor cells. However, we are nor separating CD4 from PBMCs. We really not thinking about keep or not keeping CD4 in the culture. I think when we culture PBMCs for 8-10 days in vitro probably T lymphocytes will die (I am not sure) and only monocyte derived dendritic cells will grow and differentiate. If you want to keep CD4 out of equation you can isolate PBMCs and let monocyte to adhere to plate 4-6 hrs or overnight. next day collect all non adherent cells which contain all cells except monocytes (monocytes adhere to plate). then develop these adherent monocyte to dendritic cells and that nonadherent cells (mostly contain lymphocytes) culture in the presence of IL-2 until you develop mature dendritic cells. you can separate CD4 from those lymphocyte population and co-culture with mature dendritic cells. I never did CD4 and dendritic cell cultures. I only worked on pure dendritic cells.
Thank you Mr. Vijay , We are purchasing our cell lines- so I think the best would be for me to purchase PBMC and coculture them with my cell of interest and check its immunomodulatory and immunosuppressive properties. Thanks again
And for co culturing using dynabeads cd3/cd28- cfse FACS- would using a normal magnet be sufficient to remove the beads?
Vijayamahantesh Bajegoudar as monocytes adhere to the plates, is it possible to collect the monocytes just by pipetting, or do we need to trysinze because we can observe that after culturing the BM-derived monocytes for 7 days, pipetting itself could not separate the adherent monocytes from the plates completely.
Donia Alson
You can not collect good number of monocytes just by pipetting.
Trypsinization is good idea.
If you are preparing monocytes for quantification of surface receptor expression by western blot or Flow cytometry, then trypsinization may not be good idea.
For flow cytometry purpose (for surface receptor expression), we harvest the adherent cells by PBS freeze-thaw method.
For PBS free-thaw method. Prepare calcium free 1X PBS and add 5 mM EDTA to it. Keep the PBS in free or on ice till it become ice-cold. For harvesting cells, remove culture media and immediately add 1-2 ml ice-cold PBS and incubate on ice for 10-15 min. Collect the cells by pipetting, add warm (37 degree) PBS and collect the cells by pipetting.
Vijayamahantesh Bajegoudar I am preparing the BM-derived Dendritic cells for flow cytometry so this protocol might help me. Is it a good idea to collect both floating and adherent cells for the Dendritic cell analysis. Thank you.
I am planning to assess immunogenic and immunomodulatory properties of my cell.
Does anyone have articles or recommendations..on how to go about the same.
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