I'll work with SH-SY5Y cells to evaluate the citotoxicity of a plant extract using the MTT assay to assess cell viability. I know that the cells need more FBS (20-30%) to recover from freezing, but after recovery, how much FBS should I add to the media used in the cell culture?
I am also confused about FBS% for seeding and treatment. I've searched for this information in the literature and I've found different conditions:
* 10% FBS for both seeding and treatment
* 10% FBS for seeding and 0.5% or even 0.1% FBS 1 day before treatment
* 15% FBS for seeding and 5% FBS for treatment, etc.
Moreover, I haven't yet decided the cell density for the MTT assay because I've found different informations on the literature as well, so I'm planning on performing a preliminary experiment to determine the appropriate initial seeding density. I know that FBS affects cell growth and the results of MTT assays can be affect by the number of cells, so I'm not sure about how much FBS I should add to the media in each phase of the experiments.
I hope you can help me figure out what to do. Thank you!
Hi,
For revival and seeding 10% FBS is required.
For MTT assay, no serum is used, since it will interfere with phenol red and affect the final reading.
Thanks
Nisha
Hi,
Re:
I am also confused about FBS% for seeding and treatment. I've searched for this information in the literature and I've found different conditions:
* 10% FBS for both seeding and treatment
* 10% FBS for seeding and 0.5% or even 0.1% FBS 1 day before treatment
10% FBS is a normal condition for cells. The lower percentage the day before treatment is to serum-starve your cells so that they will react 'better' especially if you're looking at phosphorylated proteins or immune activity. Often if you don't serum starve the cells, you won't find changes between your controls and your stressed/treated cells. Just so you know in the future. MTT assays are usually pretty good about telling you the conditions required, depending on the kit you're using. Good luck.
Use DMEM F-12 Ham media with 10% FBS and cell will remain healthy for long time.
Thank you very much for your help! I've talked to some of my collegues in the lab and I've decided to follow their protocol, which do not include serum starvation (15% FBS for seeding and 5% FBS for treatment). And for the MTT assay, the MTT will be diluted in serum-free DMEM. Thanks everyone.
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