I am using BRDU differentiated IMR32 neurites to find out whether my plant extract protects the neurites against amyloid peptide toxicity. Aggregation of amyloid peptide 1-42 was done in Tris-HCL ph 6 (for a week). However the amyloid did not reduce the viability of neurites substantially and was not consistent in each batch (ranging from 60% to 80%) at 0.5mg/ml concentration after 24hrs. What is the best pH for aggregation? Does it need any metals like Cu or Zn to be added in order to increase the toxicity, or do we need longer time for treatment to see cell death? Any ideas?
Hi Theanmalar.. Refer to this page. There are plenty of protocols dealing with answer to your question.
https://www.researchgate.net/project/Amyloid_open_protocols/
Good luck
Do you know for sure that your starting material was actually monomeric? If it was already fibrillar, letting it incubate for a week won't help. I always used to solubilize my Abeta in HFIP to make stocks that would be monomeric from which to begin my experiments. A good way to check the size distribution of aggregates is to perform size exclusion chromatography. This is helpful in defining what exactly is going onto your cells by fractionating the peaks into monomer, oligomer, and fibril forms. Of course Abeta is always changing, but this is a pretty good approach if you work quickly and get the fractions onto your cells.
In my work with fibrillizing Abeta I dfound that the speed of mixing played a big role in the fibril formation. I was using a tube rotator for 1.5ml tubes for my interactions. I noticed variations from batch to batch and figured out that the speed setting wasn't always consistent. Sticking with an exact speed every time substantially reduced the variation. I tracked the fibril formation using a thioflavin-T assay. There are some methods in a few of my papers, although I never actually published any cell culture experiments of the type you are doing. I'll try to get the full text up.
Another problem that you might be having is that fibrils aren't the toxic species. There is considerable debate about this, but many report that it is actually the intermediate oligomeric forms that are toxic. Perhaps you are actually incubating too long?
Aggregating AB42 for a week at low pH forms a high percentage of non-toxic fibers.
To get higher toxicity you need incomplete aggregation to fibers. The "oligomer prep" listed here is commonly used to give species with a much higher toxicity. Other preps can be found at the Researchgate project Rajesh suggested.
Whoops strike that - I treated with HFIP before lyophilyzing. I solubilized in DMSO to make stocks. Sorry, it's been a while!
- Badges
- Science topic