i wanted to screen several herbs for their potential efficacy as a drug against Alzheimer disease . I am using SH-SY5Y neuroblastoma cell lines as a model for Alzheimer. i wanted to know the details of all the methods which i should follow to completely evaluate the invitro efficacy of the herbs as a potential drug for Alzheimer before proceeding with further invivo studies on Alzheimer.
You need first to ask the question what are you expecting from the SH-SY5Y cells as AD model? As in AD you will expect neurodegeneration, and SH-SY5Y cells won't degenerate spontaneously. So, first you need to have a trigger that will couse them to degenerate as in AD cells showing Abeta accumulation and hyperphosphorylation of Tau proteins. Than you can use two approaches or rescue or of prevention which means one is treatment after degeneration starts and one is before. You can measure in a time course the cell geneneration and cell survival for example on the xCelligence which measure the cell resistance in life in your incubator, since you do not know when things happen. MTT test or other methods testing cell death and survival are dependent on a time you choose which may be wrong (you miss a lot of data).
Thanks Edna Grunblatt. i want to focus on evaluating the efficiency of herbs for rescue approaches using SH-SY5Y cells as AD model .I have 2 herbs for screening one is effective in reducing amyloid beta accumulation while the other targets tau hyperphosphoyrlation. As you have mentioned that the choice of methods depend on time which methods would be more suitable testing the efficiency of herbs in reducing amyloid beta accumulation and tau hyperphosphorylation in a rescue approach. Secondly I wanted to know which trigger would be the most suitable for making cells degenerate as in AD cells in SH-SY5Y cells line.
If you talk about rescue, usually you will start setting the experiment so that the cells death should come to at least 50% cell death, and than start to treat the cells with your herbs in various doses (from zero to the higher dose) and see whether one treatment is enought or a repeated treatment of these different doses is needed every e.g. 24h or 12h or else.
For the cell death, as SH-SY5Y won't produce Abeta plaques themselves you can use external produced toxic aggregates of Abeta (look into the literature what others use and at what conc.) and treat the cells with it to the point you receive 50% cell death. You need also to see at what time point, and best is for example using xCELLigence equipment that measure impedance at real-time and monitor cell growth/death/change of morphology. In this way you won't loose valuable information. You can also use oxidative stress approach, using ROS to get cell death. After that you can start rescue treatment and again as above look into the time response as well.
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