I would like to generate bone marrow-derived dendritic cells. Following the protocols, it seemed my cultures have a lot of different cell populations of various sizes. I am not sure if I am getting the expected results. Attached here is the photo of my Day 1 culture:
400x Magnification
I hope you could help me with this. Thanks a lot.
The bone marrow has a lot of differentiating cell types. I recommend to use CD-oriented sorting with a FACS to enrich your population after having a suspension of bone morrow cells. Hope this help.
Use a tissue culture plate one day 1 initially then remove suspensionand leave everything that sticks to the tissue culture plate behind. Propagate the progenerators in cytokines for a week or so. The main thing is letting all the mature myeloid cells, fibroblasts, and junk stick (bc it will) and the DC progenitors wont.
-simple & free way to purify the profenitor population ;)....old school...but it's soooo cool! Haha
Thank you very much for your responses. I would like to sort for DCs using CD11c magnetic beads from Miltenyi. However, we have no available kit yet. Moreover, there were plenty of protocols and articles suggesting successful generation of BMDCs so I guess I have to try those protocols first and show my data. Hopefully I'd be able to convince and justify the use of sorting beads. I am just wondering too, because cell sorting is antibody-based, cell population is more or less pure but could there be some activation during sorting?
Thank you Luke. I will try your suggestion in my next experiment. I am plating my cells in polystyrene petridish based on literatures. I am adding fresh media on day 2 or 3 while obtaining half of existing media, pellet the cells and resuspend in fresh culture medium before placing back to cells on day 4 or 5.
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