Hi,
I am a complete beginner with the worm model in non-nematode environment, thus I badly need some help. Thank you already in advance, I really do appreciate it! Namely, my problem is that I would like to perform an acute oxidative stress assay using hydrogen peroxide as a stressor to evaluate potential protective anti-oxidant effect of my plant polyphenolic compounds.
I found out that some groups use 3 mM H2O2 treatment for 5h either in liquid medium or H2O2 added to the NGM plates before checking the viability. Well, can somebody explain to me what is the advantage of one over other (liquid vs plate-based assay)? And do the animals have access to food source during these 5h of incubation? And after washing the worms should I just put them in a normal NGM plate and score alive vs paralyzed/dead animals?
Besides, if I would like to quantify the expression of oxidative stress genes between conditions (e.g. sod-2, ctl-2, hsp16.2, gpx) should it be sufficient to treat the worms for example 2h? I would be very happy for any useful references.
As for the plate vs. liquid assay, I guess it depends upon your aim…that is, is it a dead/alive assay or ‘what developmental stage do the worms reach’ assay?
Also, what’s the mw of your plant compounds, absorption into plates from surface spreading (if your compounds cannot be mixed with agar) becomes limiting if the compounds are high mw…plus, if you have small amounts then getting a good concentration range might be impossible or expensive.
Pros of liquid assay is the throughput…plates are how the worms are usually grown…you can hybrid and first expose the worms in liquid culture then allow them to develop on Op50 plates…that kinda answers your second question regarding food…they are usually fed afterwards as the assay is normally short.
As for the time course for oxidative stress genes expression profiles…that’s the wonder of science…if it’s not in the literature then you have a window of opportunity…do the experiment!
Hi Steve,
Thank you! I am glad to see that other people are also wondering how to perform this classical ox stress assay. My aim is dead/alive readout assay (treatment L1 until late L4->H2O2 stress->dead/alive count). Probably I should avoid using H2O2 co-treatments with my extract as you mentioned that H2O2 might damage them like possible SYTOX dye.
I am not sure about the compound size(s) in my extracts. Hereby I take an advantage to ask about something I have been puzzled for a while. Namely, is it known how the compounds mixed with NGM enter into worms? Is it thought mainly via diffusion through cuticle? Why the compounds are not just mixed with bacteria and it would be easy to imagine that the worms will eat bacteria/compounds and not just crawl on top of them (agar or surface spreading).
Thank you once more, Sirle
You say you want a dead/alive assay, are you interested in;
a. absolute numbers of dead or alive after ‘x’ hours/days or;
b. survival rates (Kaplan Meier curves)?
You mention wanting to treat from L1 until late L4 with H2O2, or am I reading that incorrectly?? If you really wanted to expose the worms to H2O2 over such an extended period (and I’m not sure why you would want to), then liquid culture is really the only way to ensure a constant exposure concentration.
If you are really interested in the protective effects of your plant compounds, then they need to be administrated contemporaneously or pre-emptively;
a. contemporaneously runs the risk that the OP50 catabolise your plant compounds and reduce their effectiveness/introduce ‘noise’ into your assay, see this paper;
This paper also highlights some of the differences encountered (at least with resveratrol) administering drugs and other compounds via different routes (liquid culture, alive or dead OP50 + medium, spotting plates etc.)
b. pre-emptive administration might not counteract the acute effects of H2O2 exposure, you would need to test this.
As for the administration routes…dissolving in the ngm agar or incubating worms in liquid medium are favoured methods as one knows in advance what the exposure concentration is. How this equates to uptake is another matter…some compounds will diffuse through the cuticle, but most is via absorption of ingested liquid/bacteria. as already mentioned, the difference in effective test compound concentration the worms are exposed to may depend upon whther you are using dead or alive OP50 as a food source. Might be worth taking a culture of OP50 and letting it chomp on some of your compounds…run an HPLC and see what’s left/produced etc. Then at least you have a baseline.
In the above paper at least, the most effective absorportion of the test compound (and that might be important for your assay) was using heat-killed bacteria.
All of these issues make for an interesting series of experiments…
Thank you for taking your time again. To start with, I was thinking about dead/alive assay after X hours, for example 2mM H2O2 for 5h. What I meant was to grow the worms from L1-L4 with an extract (pre-emptive administration; not H2O2), and then apply oxidative stress to see whether the treated worms are more resistant compared to control worms. I am afraid that if I would apply H2O2 contemporaneously with my extracts, they might get destroyed. Does it make sense?
Another doubt what I had was the compounds administration. The publication by Zheng and co-workers was great. This shows that my initial idea to mix the compounds directly with alive OP50 (LB medium method) was the least efficient one. The most successful administration of resveratrol and FUDR was achieved as you said by “NGM dead method” (or liquid culturing), which are the most commonly used administration form used at least in case of publications using bioactive compounds. HPLC test of compound uptake would be a perfect experiment, but in a current moment I cannot effort it. My only doubt when using dead OP50 is that when I plate starved L1-s, they develop much more slowly compared to plates grown with metabolically active OP50, and also the population does not seem “healthy” and homogenous.
If you look through the literature, you see that the worms (wildtype at least) start keeling over at about 5 - 6hours at H2O2 concentrations around 2-5mM.
]The problem with a once-off dead vs. alive count in this case is that you might miss this time point…that is, you may sometimes count too early and have 100% survival for treated AND untreated. This might be a real problem given the relative asynchronicity of sychronised L1 worms. Noise in the calculations…
I would do survival curves, you then cover a number of time periods, you seehow consistent your controls are, you can see if the protective effects of your plant compounds are transient or not…it’s a much better way.
I would incubate with your compounds…then expose to H2O2 in bacteria free medium, then move to normal plates to monitor survival. This also removes your problem concerning poor growth.
But do the control expts…fine tune your assay by doing killing time courses for your selected and other H2O2 conc. together with subsequent survival of those worms alive at the end of the H2O2 exposure for each of the strains you use. Sorry if that was an obvious point…but it often gets left out.
As for the HPLC, there must be another lab in your building that could run the extracts for you…a much more cost effective solution than saving up to buy the equipment yourself…perhaps just a column or a favour necessary.