I have been trying to test the toxicity of Amyloid beta oligomers on neurons by measuring ROS. I am observing that the ROS response on exposure to these oligomers is highly variable when tested even in consecutive experiments as well as with the same oligomer preparation. I tried to characterise the oligomer preparations on Tricine gels which I found to be consistent every time. I want to ask what factors can be there behind this variable response and how to reduce them. Is it also observed by other researchers working in this field?
Are you using secreted or synthetic Abeta? What concentration are you using and for how long? I've also observed inconsistencies in my experiments looking at various downstream effects of Abeta in neurons. The amyloid cascade hypothesis is controversial in general, and there are arguments against using cells treated with synthetic Abeta peptide as a model. Do you use hydrogen peroxide as a positive control in all your experiments? Is neuronal health consistent between experiments?
If you are measuring ROS purely as a marker of toxicity, it's worth trying some other methods, such as LDH or cell death assays.
It is well stablished that the length of the neurofibrils and the density of the amiloid tangles is very variable even within the same preparation. So, unless you are making your assay with the same preparation and the same animal you will be obtaining a lot of variability in your ROS testing.
Another question to answer is what kind of beta-amyloid are you using. The most commonly used is the 1-42 fragment, but since the most toxic fragment is the 25-35, it is possible that you will be obtaining more reliable results by using the last one. Indeed, being the beta-amyloid(25-35) a shorter peptide, it is possible that the length and density of neurofibrils and tangles have less variability, and your results would become more constant.
And, of course, it is the issue of the reliability of the method as our colleague correctly pointed before.
@Kai Liu: I use DCFDA based ROS detection. Yeah, my lab has previously published papers using the same reagent for this purpose and is widely accepted.
@Kseniya: I use synthetic Abeta peptide from which I prepare oligomers. I use 1 micromolar for 4 hours.
@David: I am using Abeta 1-42 peptide purchased from American Peptide Co. Yeah, so I try to use same preparation let's say for 3 consecutive experiments from the same cell line that I am using. I do not get consistent results in all the 3 consecutive experiments. Exposure to Abeta might lead to let's say 25% increase in ROS activation in one experiment, in second it might give me only 15% and in third it might give me no ROS increase. Also, an absence of ROS in one experiment doesn't negate the probability to observe ROS in next one. This is the level of inconsistency that I am observing. I have been doing these experiments since 6 months.
can you post or give a reference to the oligomer prep and ROS procedure you are using?
@Apurva: this kind of inconsistency that you are observing might be due to the length of your peptide. Oligomerization of a 42-mer peptide is much more variable than that of a 11-mer peptide, so the pathological effect over the ROS production might be biased for these oligomerization issues.
Consider to change to the Beta-amyloid(25-35).
@Veronica: That's a good idea... I tried out different concentrations of Abeta ranging from 1uM, 2uM and 5uM for different time points such as 4h, 6h or 12h. I found that 1uM for 4hr gives good ROS. But the main problem is with its consistency in being reproduced every time.
Last day one of my colleagues also suggested that DMSO might act as antioxidant in this case... but the DMSO concentration final is 0.1%. Will it act that way even at this concentration?
I would treat for longer. We do 24-48hr treatments. The cells don't die at 1uM but we see biochemical changes.
Are you using phenol red free media? Phenol red may interact with Abeta and impede your results
I am trying to check for ROS at 4hr or at earlier stages because oxidative stress due to Abeta is an early event rather than a much later stage event. I understand that for biochemical changes to be detected one might need to look at longer time exposures.
Maybe try changing assays, looking at RNS too, NAD+ and other metabolic markers of stress, as well as changes in some of the effectors of stress.
Some things to try assuming the problem is the oligomer prep and not the ROS assay
1. The HFIP may not be fully removed with the procedure you listed. If the residual HFIP is not completely removed (below 0.1%) it can cause numerous artifacts. Consider drying the film under a vacuum or in a dessicator for 24 hours first. Lyophillization is generally our preferred option, but i dont know how it will affect the formation of those particular oligomers.
http://www.ncbi.nlm.nih.gov/pubmed/19526294
2. If you can check the preps first with a conformation specific and general Abeta western blot before running this may narrow down the problem. The Tris gels can be deceptive as the protein oligomers are fragmented by them even under native conditions.
3. Consider switching PBS for F12 media. The response from F12 is supposedly more regular than with PBS. We have not had good luck using PBS for consistent oligomer preps (unfortunately F12 cant be used with NMR)
http://www.ncbi.nlm.nih.gov/pubmed/12499373
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