culture

Model Organism: Worms Methods & Reagents
1

hi all

i culture the c.elegans in solid NGM plates,usually.
As positive control group,i use 0.1mol/l rapamycin(DMSO as solvent ) to add the NGM(final concentration 0.0001mol/l).But the result is bad for extend lifespan.
In addition, I observe that apamycin was separated out in NGM.
How should I improve，please?
Thank u

2

Help Out，thank you.

3

buoyed up immensely by Eric’s support (it was support wasn’t it Eric?) and being critically short of glucose this evening and critically full of a great German beer, I caved in and decided to answer your post:

So, first with what is visible from your post:

  1. Your stock conc of rapamycin is ok…but in DMSO it does precipitate out and needs resuspending before use.

  2. Your working conc. of 100µmoldm-3 (or for those without a µ, 0.0001mol/l)is also in line with other work.

Now to the $64,000 stuff…

  1. ‘bad for extend lifespan’…what strain are we talking about here and what’s the difference between your test strain and control N2 in the survival curve?

  2. I nearly didn’t ask this, but when do you add your rapamycin to the agar (I assume you mean agar from your first sentence?), before or after autoclaving?

  3. If it’s after autoclaving (let’s stay optimistic), at what temperature do you add the rapamycin (hint: it is not thermostable)?

  4. How do know that it is rapamycin that is precipitating out?

  5. Where do you see the precipitate and what does it look like.

Steve

4

Firstly, many thanks to your answer.
1Compared with control ,rapamycin doesn’t extend lifespan.i both use N2
2 Yeah, after autoclaving
3 Rapamycin was added When the molten agar is 40-50 degree Centigrade.
4 I can see the precipitate clearly.they seem like white-floc
So, i am unsure what causes result in rapamycin doesn’t extend lifespan.
I’m looking forward to your help.thank you
"buoyed up immensely by Eric’s support (it was support wasn’t it Eric?) and being critically short of glucose this evening and critically full of a great German beer"What is the means?

5

If you are not observing a lifespan extension in elegans with rapamycin, you should contact Keith Blackwell and try to figure out whether there is some important difference in the way you are doing the experiment, compared to what he’s previously published.

6

I agree with Hillel, in the end if you see no difference between rapamycin-treated and control worms in terms of lifespan, then it might be a good idea to contact Keith Blackwell.

The paper linked by Hillel and the paper it refers to (Wang et al., 2010 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2916858/) for the lifespan analysis, suggests to me that their experimental setup involved exposing 1 day-old adults to 100µM rapamycin. What is your experimental setup?

It could be that the precipitate you see is unrelated to the addition of rapamycin, are you using NGM plates and adding the cholesterol, CaCl2 and MgSO4 also after autoclaving and at the appropriate temperature??

What does the quote mean…it means I was happy to answer your post.

Steve

7

Thanks again for your answer
Yeah,i choose L4 for lifespan analysis.I also use he cholesterol, CaCl2 and MgSO4 after autoclaving and adding them to agar in 40-50 degree Centigrade
So,I dont know what exactly is wrong with it?

8

I guess you assume rapamycin treatment can extend life span through targeting mTOR.
However, it may be not efficient in C. elegans, as reported by Xiaomeng Long et al “C. elegans is insensitive to rapamycin;”.
Long X, Spycher C, Han Z S, et al. TOR Deficiency in< i> C. elegans Causes Developmental Arrest and Intestinal Atrophy by Inhibition of mRNA Translation[J]. Current biology, 2002, 12(17): 1448-1461.