Dear all,
I am currently struggling trying to measure ROS in my guard cell enriched samples.
My samples are produced by blending Arabidopsis leaves and then retrieve in a 210 μm nylon mesh epidermal fragments (https://bio-protocol.org/e2447).
I would like to see the ROS response after flg22 treatment adapting the well known protocol used for leaf discs ( Article Pattern-Triggered Oxidative Burst and Seedling Growth Inhibi...
).However, I failed all the times. I tried by adding more elicitor, by not incubating overnight and by adding more epidermal fragments than the respectively amount of leaf disc.
Does anyone tried something similar before? Or any idea? No protocols are currently available for the purpose.
Hey Lucia,
I am not an expert in the field of plant cell ROS or whole tissue ROS measurements. If you have a suspension of separated guard cells after the mesh in your wells then it should be fine. If you have, so to speak, clumps of leave disc tissue it might be to bulky. Luminol is freely diffusable, but the HRP, as an protein, is not. And the HRP is critically needed for the reaction, because otherwise no light is generated, which you could measure.
The medium for cultivation could also cause a problem. If you want to measure luminescence or flurescence it must not contain any kind of pH marker (e.g. phenol red), because this quenches and/or falsifies your ROS signal. So always use clear full medium for incubation of your guard cell suspensions.
And of course it could be that your stimulus (Flagellin) in this setting just does not induce ROS production in your guard cells. Therefore a positive control (not H2O2) might be helpful. We are using for example PMA for Macrophages as a positive control. Any kind of chemical or physiological stimulus that induces ROS production in plant cells is fine.
These are just some suggestions. If you write me in detail how your experimental setup looks, perhaps we can find a solution.
All the best and stay healthy,
Marc
Hey Lucia,
I understand the difficulty in getting functional guard cells. The protoplasts are the nearest to a real single plant cell, but indeed it is not a guard cell in the normal enviroment.
The supernatant will not help. I hear often from people that ROS are "secreted" but this is completely wrong. H2O2, which is the most stable molecule, diffuses only a few micrometers. ROS are to short-lived, so your basic experimental setup is good. The point is to get separated functional guard cells. I would give it a shot with isolated protoplasts. To be honest, I think the ROS response after a pathogenic stimulus will be very quick (30 mins, 1 hour or even quicker). So it does not matter, if they are short lived. No normal ex vivo cell takes 24 hours to produce ROS after a stimulus in significant amounts. Of course it depends on the stimulus and the cell.
We measure ROS (with macrophages though) always in a plate reader and have a detailed protocol for it. Basically you do it as follows:
1) Seed your cells (between 50.000 - 100.000 cells per well) in technical triplicates. Be sure that you choose a plate that garantuees cell adherence. 2) Let them sit overnight in a full medium of your choice. 3) Centrifuge at a appropoiate speed for your cells (for macrophages it is 650g, but every cell is different), 5 mins at 4°C. 4) Remove your medium and add 100 µl of clear medium to wash away overnight medium residues (wash with the medium, which you will use for meausrement) You cannot use medium with any kind of serum or pH indicator (for example phenol red). 5) Centrifuge again 6) Remove the washing medium 7) Add 100 µl medium in wells for untreated cells 8) Add 75 µl medium in wells, which will recive the stimulus of your choice (e.g. flg22 in different concentrations) 9) Add any kind of stimulus and/or subtances in a volume of 25 µl to the 75 µl. 10) Add to every well 100 µl of your Luminol/HRP solution (the final volume will be 200 µl) 11) Place the plate in a pre-heated plate reader and measure luminscence every minute for 1-2 hours.
If you have any kind of questions please feel free to ask. And if it works it would be nice, if you site our paper for the ROS meausrement protocoll :)
I have also added an example plate scheme for you.
All the best and stay healthy, Marc
Please keep me up to date. All the best,
Marc
In context of this topic, I would like to bring to your attention a recent review of our group, "Functions of ROS in macrophages and antimicrobial immunity".
In this review, we give an introduction to ROS and their sources in macrophages, summarize the versatile roles of ROS in direct and indirect antimicrobial immune defense and provide an overview of commonly used ROS probes, ROS source inhibitors and ROS scavengers (also the difference between ROS scavengers and antioxidants, which are not synonymous, is explained).
If you like, please have a look at:
Functions of ROS in Macrophages and Antimicrobial Immunity
February 2021, Antioxidants 10(2):313
DOI: https://doi.org/10.3390/antiox10020313
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining