Hi everyone,
I am trying to generate Dendritic cells from mouse bone marrow where I am culturing the bone marrow cells with GM-CSF for 6 days and measuring the percent DCs generated on 6th day by looking for CD11c positive cells by flow cytometry. But the yield I am getting of DCs is only 60-70%.Is there any other method of DC development from mouse bone arrow which can give better yield? Should I use GM-CSF+IL-4 instead of only GM-CSF for culturing mouse bone marrow cells?
Dear Ashwini Dhamanage
Please check the attached protocols. I would suggest to add IL-4 to your culture to increase your dendritic cell differentiation. In addition, I would suggest that you increase your culture time to 10 days as that might increase your CD11c expression by 15 to 20%.
Article Generation of Bone Marrow Derived Murine Dendritic Cells for...
https://www.medscimonit.com/download/index/idArt/896951
All the best & good luck with your experiment,
Michael
HI Ashwini
It sounds like you are doing everything correctly and after 6 days you would expect that percent yield. We use the Lutz et al method (which uses GMCSF only). We routinely wait until days 8 -10 before we perform experiments - this is because by day 9 we found that you get a larger percent of cd11c + cells (approx 90%).
hope this helps
cheers
Sean
I would use recent knowledge about DL1-Notch2 signaling to get better quality DCs in vitro. See Boris Reizis's work here: https://www.cell.com/cell-reports/fulltext/S2211-1247(18)30832-5
Thank you so much guys..will look into the protocols suggested by all and get back to you in a short time..thanks again
Hello, I have gone through the mentioned protocols..@Samarth, the protocol is suitable for the generation of cDC1 types and not for cDC2 type, the cells in which I am interested..kindly give additional information in this regard..
@ Michael, the protocol mentions the replacement of medium after 12 hrs by discarding floating cells and adding fresh medium with GM-CSF and IL-6, shouldn't it be IL-4 instead of IL-6? and by discarding floating cells after 12 hrs, don't we loose pre-DCs?
Please correct me..thank you
Dear Ashwini Dhamanage
I have followed the paper in JOVE, which only used GM-CSF and IL-4 in its primary DC media, so in fact I can't speak for the switch to IL-6 in the second one. IL-6 is known to maintain immature DC state in vivo, but what the exact effect or difference between the two culture methods is, I have to plead ignorance on that one.
Article Generation of Bone Marrow Derived Murine Dendritic Cells for...
The discarding step for the floating cells is just a brief clean up to get rid of dead cells and yes probably some precursors as well. Again you would have to FACS the discarded cells to know for sure. The main consideration for which protocol you ultimately use depends a bit on your downstream application after the 10 days of culture. IL-4 for example maintains the immature state of DCs and TNFa (in the secondary media from day 6 onwards is used to induce DC maturation) for antigen dependent T cell stimulation experiments (which was my interest at the time!). If you wanna study other aspects of DC biology, I would advise to check the different culture methods side by side and chose the one most suitable for your particular application/ downstream assay.
I hope that helps at least a bit.
All the best and good luck with your experiments,
Michael
Thank you so much Michael for the information..I will definitely modify the protocol accordingly..
After getting DCs, is CD11c marker sufficient for their identification or any other markers are necessary for their purity check..how can I identify mouse macrophages contamination in extracted DC population? because there may be CD11c+ macrophage population in culture.
Hi Sean,
As recommended by you, I used Lutz M protocol for DC generation by using GM-CSF only and it worked very well, as I got >90% CD11c population at day 10, but the culture also contained high number (>75%) of CD115+ population (macrophage precursors) and after analysis I found that CD115 was present on most of the CD11c+ cells (>72%)..what could be the explanation for this kind of result? Do dendritic cells express CD115 as well?
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