I want to do drug screening on β-amyloid treated primary cultured neuron, but the toxicity on primary cultured neuron is not very obvious and cannot be easily detected by MTS. Sometimes, even stimulate the viability of the neuron. I used HFIP to dissolve β-amyloid, then followed by DMSO. I wonder whether the preparation of β-amyloid would be a problem? Since I saw many experts in this field suggested to use NH4OH or NaOH to dissolve β-amyloid.
Hi, Lisa!
I worked with Abeta a few years ago, and my first advice is follow the supplier's suggestions as how to prepare the peptide.
I think DMSO is the most common suggestion. So, you can first try to dissolve it in concentrated DMSO solution, then in cell medium.
Another important aspect is the supplier: some have more reproducible batches than others. At one point I tried 2 different suppliers in the same primary neuronal cultures and saw no decrease in viability with one of them. The other was ok (about 20%-25% decrease in MTT assay, if I recall correctly). Anecdotally, I once prepared the Abeta peptide following the supplier's instructions (dissolve in water) just to find out that the datasheet was incorrect!
Finally, there is a big difference between the soluble peptide and the aggregated one. Be sure to use the appropriate concentration and check for aggregation state.
In our lab we used soluble Abeta(1-42) and could induce toxicity in vivo and in vitro. (I am sending a reference in attachment)
Good luck!
Some of the amyloid beta peptide can not form oligomers. So monomer Abeta will not show too much toxicity. You need double check your abeta.
Hi Lisa,
abeta aggregation state can be a problem. It is shown that oligomeric form of abeta is the most neurotoxic, however, you won't see effects on cell viability at low abeta concentrations. This is probably because oligomers (nuclei) comprise only a minor population of species in the aggregating mixture. You would need to characterize your prep for the presence of aggregates (there is a lot of literature published on this subject)
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies