HUVECs are being starved for 24 hr (in 96-well) and same volume to of DCFDA is being added directly.
Do I need to replace the media before adding DCFDA?
Can cytokines cause any effect on ROS generation here? if I don't change the media?
Best
Hi Khan,
depending on the medium you use for your HUVECs you will have to change the medium (probably containing FBS) to a medium that is FBS-free since, as far as I know, the FBS contains enzymes called esterases that will 'activate' your DCF even without ROS, and would give you a false positive result.
In my experience it's also very helpful to add a positive control, such as cells treated with H2O2 that will definitely give you a strong DCF signal (if you have spare cells for this).
Since the DCF treatment itself only takes about an hour or so I don't think 'removal' of any cytokines when you change the medium would have a (strong) effect on the cells.
Whatever effects potentially produced cytokines would have on your cells, they will have had them already before your DCF treatment.
We recently switched from using DCF to using an agent called CellROX, which comes in different flavors with different spectral properties. It might be worthwhile to check those out as well. Here's a link for the 'green' version.
https://www.thermofisher.com/order/catalog/product/C10444
Hope this helps.
Dear Eric,
Thank you so much for your detail info and for the link as well. Actually, I keep my HUVEC serum-starved for 24hr and then add DCFDA (20uM) for 30min. And after that I treat the cells with Angiopoietin-1 (50ng/ml) along with my compound.
I wasn't quite sure about the idea that after 24hr is it an important thing to aspirate the media from the well before adding DCFDA.
Thank you again.
Taufiq
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