I cannot get my AB-42 oligomers to kill N2a cells plated at around 40% confluence in serum free media during treatment with oligomers. We originally dissolved the AB-42 film in HAMS F12, but we aren't in a position to order it and are looking for other options. PBS has not worked and I'm waiting on the results of the DMEM now.
Getting the F12 buffer will be much cheaper in the end then trying to redo the assay.
For an unknown reason, ABeta forms oligomers better in F12 than either DMEM or PBS buffer (see bottom of page 11618 on the page below). Tris is known to block ABeta ion channels ( J. Biol Chem, 273, 13379-13382) and also binds the Abeta monomer (our unpublished results).
The pH of 6.5 may be a problem, as protonation of the histidine (~pH 7) favors fibers formation over oligomer formation (paper below).
Do you think is worthy to try with Tris-HCl (aka Trizma hydrochloride)?
We have used DMSO as carrier for a number of neurotoxins to study mechanisns of neurotoxicity in differentiating N2a cells.
The amyloid peptide should be soluble in DMSO, which can be added to N2a cultures up to a final concentration of 0.5% v/v with no adverse effects. You just need to make sure that the non peptide treated controls also contain DMSO at the same final concentration.
I would store the DMSO peptide stocks in aliquots at -20 C or less, thaw completely and vortex-mix before use. It is also a good idea to pre-heat the medium to 37 degrees C and add peptide to the required final concentration just prior to its application, minimizing the time that cells are not kept at 37 C in their normal growth conditions.
You may also find the paper of Rushworth et al (2013;doi: 10.1074/jbc.M112.400358)
of use if you haven't already seen it. They use a 2 step organic solvent resuspension procedure (recommenede by some suppliers of the peptide) before mixing DMSO solubiilised amyloid peptide with growth medium.
I hope this helps
Alan Hargreaves
Dear Jetaime,
Tris-HCl will not kill the cells. It is the current component of the TBS buffer. When solubilized directly in TBS, the Abeta-42 will aggregate almost instantly.
DMSO is an option to completely solubilize Abeta-42, but it may prevent the amyloid oligomerization, which contributes to the cell toxicity. The amyloid could be first solubilized in DMSO, but you have to promote its aggregation under high salt conditions. Please find in the attachment a paper that describes a wide range of preparation protocols, which allow the amyloid aggregation in several steps.
Best regards,
Carmen
Dear Jetaime,
Tris-HCl will not kill the cells. It is the current component of the TBS buffer. When solubilized directly in TBS, the Abeta-42 will aggregate almost instantly.
DMSO is an option to completely solubilize Abeta-42, but it may prevent the amyloid oligomerization, which contributes to the cell toxicity. The amyloid could be first solubilized in DMSO, but you have to promote its aggregation under high salt conditions. Please find in the attachment a paper that describes a wide range of preparation protocols, which allow the amyloid aggregation in several steps.
Best regards,
Carmen
Getting the F12 buffer will be much cheaper in the end then trying to redo the assay.
For an unknown reason, ABeta forms oligomers better in F12 than either DMEM or PBS buffer (see bottom of page 11618 on the page below). Tris is known to block ABeta ion channels ( J. Biol Chem, 273, 13379-13382) and also binds the Abeta monomer (our unpublished results).
The pH of 6.5 may be a problem, as protonation of the histidine (~pH 7) favors fibers formation over oligomer formation (paper below).
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques