Hello , I am trying to identify the cytotoxicity of glycating agents on Suspension cell line. I have tried 3 to 4 times and treat glycating agent with suspension cell line followed by plating cell on the same day on the same time. And i have found irrelevant results , like cell proliferation in every concentration of glycating agents.
Your kind suggestions will be highly beneficial for me.
Thank you
Hi nimra. Regarding your experiment, do you mean you treat your cells and then you plate them? not sure if this is required for your compounds, but usually you plate your cells first, leave them for some time to attach (in case of adherent cells) and then treat them. Suspension cells are a bit different as they don’t attach, so as soon as you plate them, they are “ready”. Still, i worked with thp1 before, and after counting them, plate them all with the same cell density in all your cobdition, leave them for a few hours or overnight to sink a bit in your media and then go ahead with your treatments. If you treat yor cells first and then plate them you may have errors in between your conditions? Not sure how you plate them but if you treat your cells with a specific concentration and then you split in different wells you may dilute your compound, hence your cells are not in the same conditions. So maybe that’s why you don’t see citotoxicity. hope this helps!!
There are really no timing issue that you should worry about. The only time when timing matters is if you are testing an inhibitor to the compound. What are your time points? Are you sure that this compound is cytotoxic? It might not be for THP-1 at the concentration that you are using. Is there a reason why you are using THP-1 in suspension instead of differentiating/maturing them into adherent macrophages?
Does it is necessary to treat THP-1 with drug on the same day of seeding cell? And suggest me how to increase cell density in 75ml flask. Please suggest me the condition to grow the flask.
I want to check cell viablity.
Hi Nimra !
Hope you doing well.
If you test for cell proliferation or citotoxicity using THP-1 monocytes will be enough to test any effect on a desired drug in this case for you, glycating agents.
THP-1 are a cell suspension so that saves you a bit of time on this type of assays. 1st) See your cells at the desire concentration and 2nd) Add your compound to the wells. 3rd)After that, incubating for the amount of time is relevant for me. For example, If you wanna test the effect of glycating agetns exposure after 1,5, 10 ,12 or 24 hours. Up to you and what makes sense for your experiment and hypothesis.
Make sure to use a control for cytotoxicty ( Triton X, etc )
For subculturing ( split) THP-1 I normally splitting them at 1x10^5 cells/mL on a T75 leave it for 3/4 days and I never let me grow more then 1x10^6 cells/mL.
Anything else just message me . Glad to help !
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