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Problems with DC flow

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#1 immunologist



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Posted 13 April 2009 - 11:42 AM

I am working with dendritic cells and I am trying to determine the expression of cell surface markers IA [mouse MHC II],CD80 and CD86 using flow cytometry.
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.

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