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.
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.
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.