intracellular ROS with FACS - (Jan/10/2008 )
Hi everyone!
I try to measure intracellular ROS production using DCF-DA and Flow Cytometric analysis.My problem is that i have adherent cells.Does anyone have any idea of when should the DCF-DA should be added?Before or after thrypsinization?I have seen both techniques published.
I tried both after H2O2 treatment as positive control.when i added DCF-DA before thrypsinization i got very nice curves BUT not any MFI increase.
When i tried DCF-DA after thrypsin i had very nice MFI increase BUT i had 2 curves in each sample!Has anyone else seen that?and is it correct to measure MFI when you have 2 curves?
Any ideas?
Hi madoc!!!
I tried to mesured intracellular ROS in adherent cells by FACS and this prove. I couldn't have any nice results with treated and non-treated cells. I think that trypsinization could induce intracellular ROS production. Take care with this.
I try to measure intracellular ROS production using DCF-DA and Flow Cytometric analysis.My problem is that i have adherent cells.Does anyone have any idea of when should the DCF-DA should be added?Before or after thrypsinization?I have seen both techniques published.
I tried both after H2O2 treatment as positive control.when i added DCF-DA before thrypsinization i got very nice curves BUT not any MFI increase.
When i tried DCF-DA after thrypsin i had very nice MFI increase BUT i had 2 curves in each sample!Has anyone else seen that?and is it correct to measure MFI when you have 2 curves?
Any ideas?
I am just curious, have you checked under a fluorescence microscope to see if treated cells light up?