I've been having trouble while preparing the protocols for my assays. I want to work with undifferentiated SH-SY5Y cells to check the neurotoxicity of a plant extract. I'll use the freeze-dried extract, but I'm not sure about how to keep it sterile. I'd like to solubilize the extract in DMEM (the cell's medium) to create a stock solution with a high concentration. Then I'll make serial dilutions for the treatment.
However I don't know if I should filter the stock solution because I'm afraid it might change its original concentration. I think that sterilization in an autoclave could solve the problem. In this case, do I need to keep the medium (used as the solvent) free from FBS? Some people in my lab have told me so...
Another problem is the serum concentration. I'll seed the cells in DMEM + FBS (15%) for 24h, but for the treatment (which also lasts 24h) I'll have to replace it for DMEM + FBS (5%) to slow cell growth. If I use an autoclave to sterilize the extract solution and then make the serial dilutions in the 5% FBS medium, the FBS concentration will change, right? Do I have to correct it by adding FBS in each of the dilutions?
Autoclaving your extracts is risky, unless you can be sure that the heat will not affect the compounds in them.
And yes, if you go that route, don't autoclave with FBS—add it afterwards. Make sure you have a sham control prepared the same way!
Personally, I would go with filter sterilization. It's the simplest: dissolve your compound, filter, prep your dilutions and go.
While it is possible there could be some interactions between the filter and your compound, I think the odds of this are lower than a that of a negative thermal/heat effect. Can you assay your filtered samples to confirm the concentration?
I would make sure to consider the filter material. See for example Corning's guide:
https://www.corning.com/media/worldwide/cls/documents/CLS-FIL-004%20REV4%20DL.pdf
I would lean to a PES filter, to try to minimize binding and eliminate wetting agents.
You may be able to find a filtration device and a kit of filters that can sterilize your solutions. The one I used required that the filter setup be autoclaved before use. There were several levels of filters and a pump to drive the process. It may have come from GIBCO.
Dear Cristiane dos Santos Costa
I vote for filter sterilization, it is much more likely to affect your plant extract.
With filtering you might lose some extract ingredients but the loss will be consistent and comparable between the preps, so to some extent you can disregard it as standard error. With autoclaving you'll be denaturing many proteins hence significantly altering extract activity.
For example some toxins are heat labile --- that's why cooking is a way to make food safer (think mushrooms and beans).
I would add plant extract to a very small amount of DMEM and make a very concentrated stock, and then you can use the stock to boost your medias (the 5% FBS one and the 15% FBS one). You'll be adding the same amount of stock extract to both medias so this should not be an issue.
Please let me know if you find these suggestions useful and how it all works out.
Best,
Natasha
Thank you all for the help!
I had not considered the fact that the heat might affect the components of the medium. After reading your answers, talking to colleagues and doing some more research on how to deal with this issue, I've decided to dilute the extract in PBS and try both methods (autoclaving and filtering) to see which one works best.
Cristiane dos Santos Costa you are welcome, and I think testing both is a great idea! I am looking forward to learning what happens
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