I'm trying to measure ROS in HUVECs in a 96-well format. I started with DCF from AbCam, then tried CellROX green from Invitrogen. Despite having dose-responsive decreases in viability (MTS), and increases in necrosis (cellTOX, Promega), I see nothing in my ROS assays. Not even background. I've used the CellRox in tissue and it works great, and the CellTox assay is read at the same wavelengths as the ROX, so I know I can detect it. I just can't figure it out. Can anyone tell me what I'm missing?
Are you going to detect intracellular ROS? You can use DHE or CM2-DC_FDA, or many other dyes. You should not wash cells with any buffer befiore measure because you will have a probelm with dyes uptake. Have you tried a positive control as FDA to check dye uptake and hydrolisis inside cells? Good luck!
Thanks for the suggestions!
Jun He, I grow HUVEC to confluence in LONZA EGM-2 + 10% human serum, then treat in either no serum, or 2% serum. With the DCF, I would wash once with HBSS, then add the DCF in HBSS for 45 minutes. With the CellRox, I've tried both adding it directly to the medium as well as in HBSS for 30 minutes. I then wash 1xHBSS and read.
Taras, I've used 50nM TBHP as well as the lysis buffer that comes with the the CellTox kit as positive controls. What is FDA? I can certainly try not washing them at all, although, I think I might have even tried that once. ??
I haven't tried fixing the cells yet, as I read them right after incubating with dye. I have used the CellRox in tissue with and without fixing and both worked.
Hi, Kimberly, as a positive control I mean control for dye uptake and hydrolysis. DCF require 3 step before it detect ROS: dye uptake, it hydrolisis, and only after oxidation by ROS. FDA (Fluorescein diacetate) http://www.biomol.de/produktsuche.html?aRelated=ABD-22020 have quite similar mechanism of uptake and hydrolisis, but it consituvely fluoresent after uptake and hydrolisis. So, once you get positive FDA results, you can think about next step. Please, remember, that buffer/ medium composition have a strong impact on dye uptake. For certain cells type washing step with the buffer have a strong inhibitory effect on dye uptake. Please, also remember that DCF is not a ratiometric dye, so, you need the control of dye uptake. In particularly, you need to consider how your treatment (TbHp, for example) interact with dye uptake capacity. In some cells type TbHp may increasing dye uptake capacity, but have less effect on intracellular ROS accumulation. I do not suggest you to fix cells after labeling: dye may simple leak out from the cells because membrane .became permeable after fixation. Good luck!
how you are incubating Dye? its important the way you are incubating it? will tell what procedure you are using
Kimberly, Taras has some good suggestions. You can also try another positive control for the DCF-DA itself by adding hydrogen peroxide to your cells and see if the fluorescence increases as you would predict. Also, long load times can result in compartmentalization, which may lead to non-responsive dye. In skeletal muscle we have seen load time of 45 to 60 minutes results in loading of the dye into organelles. You may have to adjust your loading time or your time for hydrolysis of the acetate.
DHE is definitely better assay for superoxide measurement in HUVEC cells. Lucigenin assay is also good. For H2O2 measurement Amplex red assay is the best one!
To add a new potential problem to the discussion: What about the wavelength used for excitation and recorded for emission?
Your remark on "not having any background" stirrs my suspiscion that your filter set might not be ideally suited for the task.
Please check the filter characteristics (not just the peek wavelength) and compare it to the characterictics of the dyes applied.
Thank you all for your suggestions! I had no idea there were so many intricacies for this assay. I'll consider each and try them into my protocol.
Peter, I'm not sure which characteristics I should look at. The CellTox assay is detected at the same wavelengths and I get great signal from that assay on the same plate as the ROS. ??
Hi, Kimberly, DCF and FDA require exitation 480-490 nm and emission 515-530 nm. Please, also remember that in many cases extracellular ROS is even more important for cell response to stimuli, You can try DCHBS/AAP method to detect extracellular ROS. It is a good alternative to fluoresent dye. DCHBS+AAP formed a red coloration product in the presence of ROS. You can quantified ROS by this way as A510 nm. You can look original paper (Van Gestelen, 1998) http://link.springer.com/article/10.1007%2FBF01279297?LI=true#page-1
or my paper from 2007 for details. Good luck!
Concerning the characteristics: please pursuit the direction Taras Pasternak has pointed out and check out whether you can e.g. change wavelengths to improved excitation.
If I understood you correctly, you didn't get any signal from DCF? Please try another lot or borrow some from a neighboring lab.
Does anyone has a good protocl for the superoxide detection directly in an in vitro system. I have seen that several protocol provide a cellular detachment but honestly I would like to keep my cells attached to the plate and make the assay directly into the 96-well plate.
Can you give me a feedback??
Hi,
I am measuring my superoxide anion with through a lucigenin assay test, that allows to measure the luminescence directly related to the superoxide anion produced. I would like to receive confirmation about the luminescence measurement: do I need a particular filter to read the luminescence? I don't think so as I have read that is not required a filter for the luminescence detection. It's true?