Dear Yasmine,

it seems that the sensitivity of the fluorometer is not high enough for the fluorescein concentrations you use. Cuvette readers like the fluoromax are often more sensitive than e.g. plate readers. You could try to incubate more cells with your particles (e.g. multiple plates) and lyze and homogenize the cells then measure fluorescence of the lysates. If you want to quantify the fluorescence, always handle a sample of a known concentration of the particles the same way, because fluorescence in nanoparticles might be quenched because of there close proximity.

best wishes

Dear Dr Ramtin,

Thanks a lot for your answer. However, if the fluorometer is not sensitive to the fluorescence of nanoparticles up-taken by the cells, how is it sensitive to the curve of fluorescent nanoparticles in which they have range of different concentrations including concentrations lower than the minimum concentration of nanoparticles i added to the cells to be up-taken?

Dear Yasmin,

I'm not sure how you make your measurements with the fluorometer, but I guess the amount of particeles that are really taken up by the cells is lower than the amount you add at the beginning. If you replace your media before measurement, you remove the particles that are not taken up, which might be the majority. In the microscope you might still be able to detect the fluorescence because it is locally higher, e.g. in endosomes etc..

I agree with Dr. Rahmanzadeh's answers.

Have you tried manipulating the settings on your plate reader to maximize your signal? Widening the slits is of course a first step. If that makes you lose too much signal-to-noise due to scattering, you can try adjusting the filters. Black microwell plates will give you the best signal-to-noise. If your instrument has a bottom reading mode, that will also give better signal-to-noise. You can get black sided microwell plates with clear bottoms that allow you to use bottom reading mode and also allow you to conduct standard microscopy. A lot of this of course depends on the limitations of your instrument.

Pipetting the lysed cells out from the microwells and into cuvettes can be a (non-ideal and tedious) solution to weak signal.

Yasmine,

Depending on the size of your particles, they can be taken up by the cells and into lysosomes. Fluorescein is pH sensitive, and it will be "quenched" at low pH. If you lyse your cells as suggested, you may see if this is the problem. If your cells that you are using for your confocal studies are fixed, you have disrupted the lysosomal membrane, so you do not observe the pH problem with fluorescein under these conditions.