Can someone advice me as to how to design a study to reveal the biocompatibility of some iron nanoparticles on a fibroblast cell line using flow cytometry? I would like to use Anexin-FITC (FL1) and PI (FL3). How can I prepare the negative control (apoptotic cells)? How can I determine weather the nanoparticles have own fluorescence? Thank you very much in advance.
To adress the fluorescence of your nanoparticles you should run a data acquisition with all the channels of the flow cytometer using your cells incubated with the nanos, that's easy. Or you can check it in a fluorescence microscope with the different excitations.
There are a number of protocols to produce apoptotic cells, maybe the easiest is to cultivate your cells without serum: in 3-5 days they should detach due to the apoptosis triggering. Etoposide and other chemicals can also induce apoptosis.
Hi Bianca:
I agree with Santiago regarding the way to address the autofluorescence of your nanoparticles.
In order to induce apoptosis of your cells as a positive control for PS exposition, I would suggest also to try DMSO: 5-10% final concentration for 5x10(4) cells/200 ul should induce apoptosis in 48 h culture.
Remember that you cannot fix your cells if you will employ Annexin V-FITC+IP, so you will need to adquire your results immediatly after staining.
Good luck!
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