Hi Ishan,

I never worked w/ IF-imaging of nanoparticles. So I can only tell you how long a FITC-conjugated Ab is incubated w/ cells in IF. We do so for 1h at room temperature. But in case of intact cells you may need entirely differnt time. The nanoparticle may not be taken up at all. You have to find this out by using live imaging. If that is not possible a time range is required. Culture multiple coverslips w/ cells, incubate w/ the nanoparticles for various time points (e.g. 0, 15, 30, 60, 90 and 120 minutes). Then observe the results. Tip: to make the 'harvest' of coverslips easy you better work in a reverse order: at t=0 you start w/ sample "120 min", half an hour later add the nanoparticles to "90 min". In this way you can stop the incubation time w/o disturbing the other wells.

Dapi will enter the cells easily. 5-10 minutes must be enough.

Thanks, Duco Kramer. I will try this approach in my research.

Dear Ishan Sandeep Shah ,

I would recommend to switch from DAPI to Hoechst 33342 as the nuclear stain, because it is membrane permeable and will stain your cultured cells. Than you should be able to run a live cell imaging experiment (assuming that the concentration of the FITC-Ova in the medium is by far lower than in the RAW cells.

Otherwise you might also switch from an microscopy approach (which I'm assuming from your question) to a FACS analysis approach using samples which have been incubated with various time points. Therefore you should not vary the harvesting, but the addition of the FITC-Ova. Then you would be able to analyse all samples at the same time on the FACS which is by far also the easier approach to quantify the uptake afterwards. Everything from microscopic image segmentation and evaluation of fluorescent intensities in relation to your DAPI staining, will be a painful learning, if you do not have somebody with experience in the lab.

Best wishes Soenke