Loss of YFP signal in transduced T cells
Posted 24 March 2011 - 05:23 AM
I am really confused looking at the FACS readout of my transduced primary mouse T cells. I hope somebody might be of help and shed some intellectual insight on the problem.
I used PlatE packaging cells lines and a retroviral plasmid construct (which has 5'LTR promoter, gene of interest tagged to YFP, IRES and HuCD2) to produce virus particles which was used to transduce T cells. I also infected 3T3 cells with the same virus.
The FACS readout shows a good expression of HuCD2 (detected with PE) and also YFP for the 3T3 cells. However when in T cells I just have the expression of HuCD2 whereas I do not have a signal for YFP.
What could be the problem? I have checked the compensation on Caliber that does not seem to be the issue.
Does anybody know if LTR is not suitable as promoter in a T cell.
I am at complete loss to understand the results.
Any help/ ideas is deeply appreciated.
Posted 24 March 2011 - 09:34 AM
Posted 24 March 2011 - 03:14 PM
Posted 25 March 2011 - 12:03 AM
Posted 25 March 2011 - 07:32 AM
Posted 26 March 2011 - 08:51 AM
Posted 19 April 2011 - 04:34 PM
Posted 20 April 2011 - 12:04 AM
Wait, I had it somewhere...
I have used this one: http://research.stow...tract/index.htm, with which you can export your .fcs files to excel and calculate the R2 value. I think it is for free (as in beer, not as in speech).