I am testing the safety of negatively charged nandiamonds on a bladder cancer cell line HT-1197 using MTT assay.
I made 5 concentration from 50 ug/ml down to 0.005 ug/ml and all the 5 concentrations are showing viability that ranges from 81 to 84 % of the control irrespective of the concentration of the nanoparticles.
The nanpoarticles were applied for 4 h (mixed with the culture medium), removed and fresh medium was added. After 48 h, the MTT assay was performed as per Sigma protocol.
The control was made with the same procedure with culture medium added during the mentioned 4h.
Can any one explain this?
You should try this assay in triplicates for controls as well as test samples. The MTT assay depends on the absolute number of live cells, thus small variations in cell number may cause big differences in 48 h, due to cell growth.
You should also consider keeping the drug in the media for the entirety of the incubation period, to maximize the effect.
The culture should not be more than 60% confluent at the time of adding the test substance, because if you use 90% confluence there may be probably two doubling events in 48h, causing the cells to overgrow and thus also causing the cells to die, or experience contact inhibition.
We observed and it has also been reported (Patravale et al., Nanoparticulate Drug Delivery, 2012) that MTT is not the best assay for cytotoxicity test with nanoparticles. Choose some other assays such as MTS or Resazurin to obtain relevant data.
Hi Moustafa, the concentrations of the nanoparticles in solution matters less than their concentrations on the cell surface.
Any inorganic particles coating the cell surface will impede their function. Internalizing these inorganic particles will probably kill the cells.
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