I want to evaluate the citotoxicity of a plant extract in SH-SY5Y cells. For my experiments, I'll incubate the cells with different concentrations of the plant exctract for 24h and then I'll perform the MTT assay to check cell viability, but I'm not sure about the cell density when plating the cells. So after some research I've decided to make a standard curve for the MTT assay, however I don't know exactly how to do it.
I've been thinking about doing as follows:
- Cultivate cells in DMEM/F12 + 15% FBS until they reach ~80% confluence
- Plate them in different cell densities, with a series of dilutions ranging from 10^6 to 10^3 cells/mL
- Wait 24h for the cells to atach and reach ~70% confluence
- Change medium to DMEM/F12 + 5% FBS (to hold cell growth) and incubate cells for another 24h (wich will be the treatment period with the plant extract during the experiments)
- Remove medium, wash cells with PBS and add serum free DMEM/F12 + MTT 0.5 mg/mL
- Incubate for 1h (or until formazan crystals are visible in microscope)
- Remove MTT solution and dissolve formazan crystals in DMSO
- Read absorbance at 570nm.
So, do I need to perform this preliminary MTT assay using a known citotoxic chemical (such as H2O2) or should it be perfomerd without any cell injury?
As the concentrations of your chemical is increased (ideally exponentially), you should get a curve with an asymptotic approximation to a certain formazan absorption level corresponding to 100% killing. If you do not get this curve shape your drug is not efficient enough to reduce mitochondrial activity and you should add a positive control.
I would consider to switch to Resazurin, which also is used to measure mitochondrial activity. With Resazurin, you can measure your culture medium of each well, but the cells remain viable. When the samples for Resazurin are extracted, you are able to lyse the cells and you can determine total DNA content using picogreen assay. In this wat you are able to calculate the mitochondrial activity normalised to DNA content, for each well. So no need for a standard curve as you always correct for DNA content, which will relatie to the total cell number seeded in your well.
Hello! Thank you very much for answering my question!
Yusuf Hussain
I wasn't planning on doing a standard curve for the H2O2 because some of my colleagues have already recommended 300mM, but now that you have mentioned it, I think it's better to do so. I have also checked the literature and 300mM seems to be to high. Thank you for suggesting.Marcel Vlig , thank you for your suggestion. I'm afraid we don't have Resazurin in our lab and can't afford buying it right now, but I will keep that in mind for future projetcts. It sounds like a more accurate way to assess mitochondrial activity.
I have decided to do the preliminary MTT standard curve without any other toxic compound since the aim is to determine the cell density for the actual MTT assays, in which I will evaluate the citotoxicity of the plant extract I'm working with compared to a positive control (H2O2).
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