Dendritic Cells and Flow Cytometry - (Apr/07/2009 )
I am working with dendritic cells and I am trying to determine the expression of cell surface markers IA
I bought antibodies for the above mentioned markers from ebiosciences and tried it on splenocytes to check the antibodies. It worked perfectly fine. When I tried using my dendritic cells < bone marrow derived and JAWSII cell line>, initilally it did stain for IA,CD80 and CD86 but it was much lower than what was expected in Dcs stimulated with LPS.
I tried different concentrations of LPS to stimulate Dcs but it did not work. From the recent data that I have obtained, I am not getting any/ significantly low populations of IA positive /Cd80/CD86 DCs that have been stimulated with LPS. I even tried using protease inhibitors to check whether that had any effect, but it did not. Initially I used to fix the cells after staining using 10% buffered formalin but I realized that it might be too concentrated so I used 1% buffered formalin. That also did not help.
I am really frustrated because I am unable to proceed with any of my work due to this.
If anyone has any clue or idea about this , I would greatly appreciate if you could help.
Thank You.
Hi there
How long are you culturing your BM cells after isolation and in what kind of media/cytokines are you using?
Clare
Clare,
I culture them for ten days in RPMI media
immunologist on Apr 8 2009, 04:52 PM said:
I culture them for ten days in RPMI media
I'm no DC expert but when I have cultured DCs from BM I would culture them in media (KDS RPMI +2-ME +10% FCS) with Flt3-L (also for 10 days). And the FACS staining was always nice and DC-like (i.e: I used markers like CD45RA,CD11c, CD11b and HSA).
So you do not use GM-CSF at all? Are these DCs in an immature state?
immunologist on Apr 9 2009, 06:14 PM said:
Nope. Just Flt3-L as suggested by the DC lab (Ken Shortman's) at my old institute. I would get pDC as well as conventionals (both CD8+ and CD8-). That's all I looked at though.
Clare
Clare on Apr 14 2009, 12:06 PM said:
immunologist on Apr 9 2009, 06:14 PM said:
Nope. Just Flt3-L as suggested by the DC lab (Ken Shortman's) at my old institute. I would get pDC as well as conventionals (both CD8+ and CD8-). That's all I looked at though.
Clare
If I remember correctly from my DC days, BM cells in the presence of Flt3-L will differentiate mostly into plasmacytoid DCs, while BM cells in the presence of GM-CSF will differentiate mostly into myeloid DCs.
I always used GM-CSF for 10 days, and got ~90% CD11c+ cells.
Can you tell exactly what protocol you use to get DCs from BM (plates, volumes, feeding), and also how do you stimulate the cells (when, how long, again volumes, plates), and how do you stain them.
Cheers.
YAY! I knew a DC person would come along eventually
Clare
If I remember correctly from my DC days, BM cells in the presence of Flt3-L will differentiate mostly into plasmacytoid DCs, while BM cells in the presence of GM-CSF will differentiate mostly into myeloid DCs.
I always used GM-CSF for 10 days, and got ~90% CD11c+ cells.
Can you tell exactly what protocol you use to get DCs from BM (plates, volumes, feeding), and also how do you stimulate the cells (when, how long, again volumes, plates), and how do you stain them.
Cheers.