We have prepared a nanoformulation of anticancer drug and performed MTT and SRB assay of API, nanoformulation and placebo (nanoformulation without drug) at concentrations of 5-100 ug/ml.
OD readings were observed at a range of 2-3 for all the concentrations for MTT assay. We thought that MTT is interfering with the formulation components and tried with SRB assay.
In SRB assay, we found OD of placebo was more than control
For nanoformulation OD readings were also more than control but less than placebo
Can anyone suggest the interpretation of results or share any protocol for the same
Regards,
Manikanta
If you can check the concentration of your stock solution of MTT, you can dilute it to bring the values down.
Thank u Fazil. We have done the cell titration assay and optimized MTT assay with the concentration of 5 mg/ml MTT. But sure we will try by reducing the MTT concentration.
make sure you got the right controls if you are diluting the MTT solution.
i have dont tons of MTT experiments so if you have any questions feel free to ask.
I think you could perform an MTT/SRB assay in wells without cells but with treatment to verifiy if the chemical structures/activity of your formulation (API, Nano+API, Nano+placebo) interfire with the OD. Then you could work as following:
OD working = OD cells+formulation - OD formulation alone
Moreover, a basic question, have u checked the treated cells? Are they less than control?
Thank you Diego. To check the interference of formulation with MTT, we have added MTT and formulation and kept for 3 hrs incubation. We couldnt find any significant change.
We also checked the IC50 of anticancer drug and is matching with the literature.
I want to ask one more thing. Nanoformulation which we are adding to wells is in suspension form. Does it effect on Cell proliferation?
I can suggest to make the treatment, then to collect medium (with treatment), wash wells and cells with PBS and then add new fresh medium and MTT. In this way you will "delete" possible interferences with the MTT and formulation..
I suggest also to move from MTT to MTS (Promega) that no require solvent for the formazan.
We also tried to wash with PBS, because of incubation for 24-48 hrs the particles are settling on the plate and we couldnt wash the wells efficiently.
did you get any OD values? did you suspended the crystals with acidified isopropanol?
I added my publication for some tips and tricks.
regards
Johan
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