Hi Prabhjot,

Preparing samples for cytotoxicity assay could be a little tricky. For this purpose, you first want to separate out the different species. For example, if you want to study the fibrils for cytotoxicity, you either 1) use a cut-off filter to collect larger species/fibrils in the retentate fraction, or 2) carry out high speed centrifugation (ultracentrifugation, if required) and harvest the fibrils in the pellet. To use for a cytotoxicity assay, you will require to resuspend the pellet from ultracentrifugation using a suitable buffer, and doing so might cause a little dissociation of the fibrils to release relatively smaller species; however the resuspended material is clearly a better option over a solution mixture of monomers, oligomers and fibrils. In case you are planning on studying the oligomers for cytotoxicity, you will either 1) need a special separation method like size exclusion chromatography or 2) samples collected at a very early stage of aggregation, when the mature fibrils are not formed yet. If you prefer the latter approach for oligomer collection, you should first follow the morphology of species formed in solution during the course of aggregation using Transmission Electron Microscopy or Atomic Force Microscopy so that you will be sure of what material you are using for your cell toxicity assay. Having said that, none of the above described methods will help you precisely quantify what species you are dealing with, however these will definitely enrich one species over the others, and that eventually will increase the confidence level of what you are testing in a given experiment.

Hope this helps. Good luck!

Anoop