I am attempting to induce senescence in NIH3T3 cells to treat with compounds to test their hypothesized anti-senescence activity. However, I have been having some challenges. Several reviews and papers that I've perused, of which I will post two below, explain in their methods how to induce senescence in this cell line.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101959/
This review explains that ~200 uM H2O2 exposure for 2 hrs, followed by washing with fresh media, and 48 hr incubation in fresh media, and the repeating of this method twice more for a total of three times, will be sufficient prolonged H2O2 treatment to induce senescence .
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The method in this paper says to treat the NIH3T3 cells with 400 uM for 45 minutes before washing and seeding out.
I am mainly referring to the section of the review explaining Oxidative Stress-induced Senescence. First, I think your calculations for that H2O2 concentration were incorrect in step 4. Using the dilution equation M1V1 = M2V2 and the volumes provided in the review: (9.789 M)(11 uL) = M2 * (11000 uL), since the concentration of 30% H2O2 is roughly 9.789 M; this results in the concentration of H2O2 in D10 media to be ~20 mM rather than ~200 uM as the review states. I corrected for this, adding 1.02 uL of 30% H2O2 to 50 mL (50000 uL) to make a ~200 uM H2O2 solution.
It says to treat cells with ~200 uM for 2 hrs, washed them with fresh media, then re-incubate them in fresh media for 48 hrs, before repeating the method twice more for a total of three times, before treating them with any compound. However, 24 hrs after the first cycle of treatment with ~200 uM H2O2, washing with fresh media, and re-incubation in fresh media, I've found that most of my cells are floating (probably dead), so I don't know what I am doing incorrectly.
The review says to make a dose response curve to evaluate H2O2 toxicity, but other articles that I have perused have stated 100-500 uM H2O2 is sub-toxic to NIH3T3 cells, so I have been treating with 200 uM for 2 hrs as the review states.
Has anyone had trouble with senescence inductions like this?
You need to optimise the H2O2 concentrations with the cell line under your experimental condition. Our papers are not using NIH3T3 but may be helpful:
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Harald Kühnel
I will perform cytotox assays with Etoposide to determine correct concentrations to use to for senescence inductions, thank you for your advice.- Badges
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