subhchitra

I have to check the level of ros after electroporation of my gene of interest to see the difference between Ros level of untransfected cells and transfected cell with gene of interest.I have used carboxy-h2dcfda (invitrogen) to stain ros.But unexpectedly Igot higher level of ros in untransfected cells compared to transfected cells.This result is again confusing with higher level of ros in untransfected cells compared to mock transfection with null vector.

the protocol I have followed in this experiment :

-10x106 cell were transfected  by electroporation by using biorad electroporator with gene of interest and null vector .
-24hrs of post transfection,cell were harvested and washed with PBS.
-From that,one million cells were taken and mixed with RPMI (10%FBS) media.
-to that 10 micromole of c-h2dcfda was added and mixed it well.
-then the cells were incubated at 37 degree for 30 min.
-after that,cells were washed with pbs once and immediately acquired in flow cytometry.

can any one please help me out why this is happening or any one  having a different protocol for  ros staining of electroporated cells.

Any suggestion?


Thank you