Dendritic cell growth medium - (Nov/04/2008 )
I am using JAWSII cells for my experiments.Someof the refernces that I came across have used JAWSII cells and have proved that JAWSII cells can substitute for bone marrow derived dendritic cells since they behave exactly the same way as BMDCs do.
Whn I tried to stimulate JAWSII cells and drive them to maturation using LPS, my cells did not release IL12. Even CD
80 expression does not increase in comparison with the untreated control groups.I felt that they were not even becoming mature DC since some of the hallmarks of mature dc are Il12 prodcution and increased cell surface marker expression of CD80 and IA.
I realized that I have been using a different growth medium than the one described in the papers. They have used MEM with ribonucleosides and I am using RPMI medium without ribonucleosides. When I checked the medium composition, except for the ribonucleosides, there was'nt much difference between the two. Could this be a reason why my cells are not releasing IL-12?
I don't even know whether my cells are maturing or not...I was wondering whethher anyone could help me with this.
Thanks.
Why don't you just derive your own BM DCs?
Clare
Whn I tried to stimulate JAWSII cells and drive them to maturation using LPS, my cells did not release IL12. Even CD
80 expression does not increase in comparison with the untreated control groups.I felt that they were not even becoming mature DC since some of the hallmarks of mature dc are Il12 prodcution and increased cell surface marker expression of CD80 and IA.
I realized that I have been using a different growth medium than the one described in the papers. They have used MEM with ribonucleosides and I am using RPMI medium without ribonucleosides. When I checked the medium composition, except for the ribonucleosides, there was'nt much difference between the two. Could this be a reason why my cells are not releasing IL-12?
I don't even know whether my cells are maturing or not...I was wondering whethher anyone could help me with this.
Thanks.
One main reason is that the yield is not very high. I have to sacrifice a lot of mice for each of my experiments.Second, since I am planning to do RT-PCR, I need good quality RNA and I am not very sure that the cells that I isolate from bone marrow will be a pure population of cells.
Clare
Whn I tried to stimulate JAWSII cells and drive them to maturation using LPS, my cells did not release IL12. Even CD
80 expression does not increase in comparison with the untreated control groups.I felt that they were not even becoming mature DC since some of the hallmarks of mature dc are Il12 prodcution and increased cell surface marker expression of CD80 and IA.
I realized that I have been using a different growth medium than the one described in the papers. They have used MEM with ribonucleosides and I am using RPMI medium without ribonucleosides. When I checked the medium composition, except for the ribonucleosides, there was'nt much difference between the two. Could this be a reason why my cells are not releasing IL-12?
I don't even know whether my cells are maturing or not...I was wondering whethher anyone could help me with this.
Thanks.
[quote name='Immunologist' date='Nov 4 2008, 06:01 PM' post='156246']
One main reason is that the yield is not very high. I have to sacrifice a lot of mice for each of my experiments.Second, since I am planning to do RT-PCR, I need good quality RNA and I am not very sure that the cells that I isolate from bone marrow will be a pure population of cells.
You could just FACS-sort the cells. You wouldn't need *that* much RNA if it's only for RT-PCR. I would go ex vivo cells over a cell line any day.
Clare