Alternative Cytotoxicity Assays - (May/03/2012 )
Afternoon everyone. I have a quick question I was wondering if someone could give me some advise with. I'm doing a bit of collaboration work with a materials science lab that has now become interested in the toxicity of their nanoparticle formulations.
These particles are combinations of magnesium and titanium. As they slowly degrade in aquaous solutions, they begins to release hydrogen gas. Obviously H2 gas is a very potent reducing agent.
These particles are not very tightly controlled in size, so some of the particles I believe are being internalized, while larger ones are just sitting on the cell surface.
The problem I am having is any cytotoxicity assay I have been using is giving me huge false positives, again due to the reducing power of the particles. I have fixed, washed, pelleted and resuspended the cells, basically any manner I can think of to remove as many particles as possible after X time point. Lower concentrations of particles don't seem to interfere, but I can't pull together a good viability curve as the particle concentration increases obviously.
I have also tried many different assays. I've tried MTT, XTT, Acridine Orange, Alamar Blue, Calcean AM. All of these have been succeptable to false positives.
Does anyone have any ideas for alternative assays that might not interfere with these particles?
You could possibly try neutral red? Other option is to measure total protein concentration (maybe use a fluorescence based assay such as fluorescamine) and compare the quantity of protein between your non-particle exposed cells. Using t-test you could determine when you see a significant reduction in protein concentration. you need to wash the cells well and freeze them in water to lyse them prior to measuring protein.
If yu ahve access to a luminometer, I woudl suggest trying CellTiter-Glo. It shouldn't be susceptable to the reducing potential or light scattering from the particles. Feel free to contact Promega Technical Serivices if you ahve any questions or concerns.
Obviously viability assays that rely on redox activity are not good in your case. In addition to ATP content assay suggested by Kevin, you may look into Life Tech's CyQUANT® Direct Cell Proliferation Assay that quantitates total DNA in the well or Sigma's
Sulforhodamine B Assay that quantitates intact cells. But both are staining based and I am not sure if your nanoparticles would trap the dyes or not, or if the particles would trap the cell-free DNA or not. If everything fails, maybe you could try EdU or BrdU DNA incorporation based assays.
You can do DNA content assays - just remove the medium, lyse the cells and add sybr green or similar fluorescent DNA dye - read on a plate reader.