Hi everyone,
I want to use the amplex red assay to quantify H2O2 levels in different cellular clones. Many protocols I found online or previous answers on RG contradict each other.
The major debate is about performing the assay:
- on cultured cells directly
or
- on culture media removed from cell cultures
What's best in your opinion ?
Someone also said that phenol red could interfere with the assay.
Thanks for the help you can provide !
Try this
Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit
https://www.thermofisher.com/order/catalog/product/A22188
Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit
📷
Catalog number: A22188
Hi everyone,
So I was able to quantify H2O2 production from my living cells by following this protocol I found. Sorry I don’t have the article’s ref in which I found it.
- plate 300k U-2 OS cells in 6 wells plate
- the next day, remove culture medium and wash cell layer with 1X PBS
- add 0.5 mL 1X PBS on your cells and incubate for 1h
(In 6 well plates, I wouldn’t go under 500µL for 1h to avoid dry some of the cell layer)
(The goal here is to kinda concentrate ROS in the PBS, so it become easier to detect via red amplex. But remember that all cells have very different rate of ROS production. For me 1h worked best and I wouldn’t recommend to incubate longer because incubating cells in just PBS stress them out. Given your cell line, 10 min could be enough)
- take 50 µL of your incubated PBS in an opaque black 96 wells plate and add 50 µL the red amplex mix
(also work with white opaque plates, never tested with clear plastic though),
(Here I use half of the red amplex reagent recommended in the manufacturer’s protocol)
(the 6 well plate format suited me the best because I was able to use a multichannel pipette to take my 3 time 50 µL in one go and have my triplicate in the 96 well plate. ( I don’t know if I’m really clear on this one…))
- then follow manufacturer’s instruction concerning waiting time, temperature, shaking of the plate and detection.
Of course you will need to scale up and down experiment given the culture vessel, cells, etc… you are using !
By following this protocol, I was able to reproduce comparable results compared to DCF/flow cytometry.
Here is the article using DCF figure 3.A Article HPV16 E6* induces oxidative stress and DNA damage.
Here is our article using red amplex figure 3.D
Article Comparative RNA sequencing reveals that HPV16 E6 abrogates t...
In the two experiments we observe a 20% ROS increase when U-2 OS cells ectopically express the E6*I isoform from HPV16.
Dear Muhammad Asad Uz Zaman,
1. I think it will not change a thing ! I'm personally used to working with 6 well plates but as long as you scale down your number of cell to fit a 96 well it should be ok.
2. KRPG is surely a "more complete" buffer, closer to a culture medium than just PBS but It's still lack a lot of things (serum, aminoacids, other salts and ions) for cells to live long in it.
I honestly don't know if your cells are going to survive longer or not. And if they do, I don't know if they still will have a physiological ROS production past a certain amount of time.
What is sure, is that the kit will work with KRPG ! Here are some documents treating about the kit and KRPG:
https://assets.thermofisher.com/TFS-Assets/LSG/manuals/mp22188.pdf
https://s3-us-west-2.amazonaws.com/oww-files-public/6/6d/Amplex_Red_-_H2O2.pdf
https://www.biocompare.com/Product-Reviews/557858-Excellent-Amplex-Red-Hydrogen-Peroxide-Peroxidase-Assay-Kit/
https://labs.pbrc.edu/cellbiology/documents/ROXMeasurements.pdf
Article An in vitro alveolar macrophage assay for predicting the sho...
Before using the kit which is pretty expensive if I recall, you can test different time point with your cells in KRPG just to see how they behave.
Have a nice day.
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