EAT-2 (ad1116) strain issues

Hi everyone! I am currently working with the eat-2 (ad1116) strain for some aging assays I am trying to set up, and I am having the issue of not getting enough viable embryos after bleaching. I follow the standard bleaching protocol (1mL bleach + 0.5mL 5M NaOH to 3.5mL of gravid worms in water). After 3 minutes of shaking, I add 10ml of M9 directly to stop the reaction and then wash 5 more times with M9. Previously, I was starving them overnight to get a synchronized population of L1s, but I did not recover enough to do my experiment, and the second time I tried synchronizing overnight, I only got like 10 viable worms.

I recently learned that eat-2 is not supposed to be starved to synchronize, so I plated the embryos directly after bleaching and washing thoroughly with M9 onto OP50 seeded NGM plates. However, I am getting less than 1% of hatched eggs the next day and some of the eggs that have not hatched look weird and kind of dissolved.

I was wondering what is the best way to get a large amount of synchronized worms for this eat-2 strain so that I can finally do this aging assay.

I should also mention that when I do the same for my N2 strains, everything is fine and I get plenty of worms.

Any input and help is greatly appreciated!!

hi there, could you try doing a limited lay instead? Pick a bunch of gravids and let them lay eggs for 4 hours, then pick them off the plate.

Hi I use to dissolve C.elegans in 1 mL 5M NaOH + 1mL bleach making it directly upto 12 mL with M9, allowing it in shaker 7 to 8 minutes. Then centrifuging it 1800 RPM for 50 seconds, repeating washing 3-4 time with M9 buffer. See if it works, some times freshly prepared NaOH becoming toxic to eggs also, so in these conditions it is better to reduce the incubation duration.

Does your bleach assay work for N2s?

I find that different suppliers or even bottles/batches of bleach actually vary a lot for me. Rather than timing the bleach assay, I keep checking on the worms on the scope. You want to stop the treatment with M9 pretty much immediately when you see a lot of the worms starting to split at the vulva, which is like the first obvious step in dissolving and they are nearly there, and in my hands then I end up with eggs after washing etc. If I leave it until it is just eggs I find they mostly die.

I think you can probably optimise the assay for your worms/bleach by changing the time of treatment, which might help in the long run if you need to use it a lot.

If its just a lifespan though, I would synchronise them at L4 by picking L4s the day before I run the assay, so you don’t really need to bleach at all. I guess it depends if you want to “leave in” differences in how long lines take to develop or not.