The publications provided above should give you info on excitation/emission and appropriate settings. I'd first try to check whether you can detect intracellular doxo about 1 h after addition in high extracellular amounts (as some sort of positive control). In case you may have problems seeing it, make sure to use a cancer cell line that responds with apoptosis to doxo. If you observe cell death after 24-32 h you at least know that doxo went in. Compare your signals to mock treated cells.

Importantly, time-lapse imaging is extremely messy with doxo, since it is a strong photosensitizer. For the fun of it, if using confocal, run a time-course over night. You'll most likely see that all cells in the field of view will be dead. When you move to a differetn region, cell death with be much lower or absent. As it seems that you are interested in early time points, this may not be a concern.

Have you considered using cell suspensions? Not sure how good it will work, but you may be able to pick up a signal spectroscopically.

Could you expand a bit on this, Rodi, regarding the experimental context? I.e. is it a fixed time point you are interested in or rather uptake kinetics.

This paper may be a good starting point regarding settings and possible read-outs.

http://www.ncbi.nlm.nih.gov/pubmed/1451589

thank you Markus , what I wan to do is a fixed time point more than a kinetic .

Hi Rodi,

could you expand on your protocol a bit? How are you planning to visualise doxorubicin? It must be linked to something for detection.

Perhaps the following link can help.

http://ro.uow.edu.au/cgi/viewcontent.cgi?article=1919&context=scipapers

Cheers

thank you Aniko , actually Im studying the effect of ultrasound contrast agents on enhancing doxorubicin uptake .so what I want is to check the penetration of doxorubicn after treatment (5-10 minutes ) also I would like to stain the nucleous and the endosome ...and I'm not sure if thats goining to be possible in life imaging .

The publications provided above should give you info on excitation/emission and appropriate settings. I'd first try to check whether you can detect intracellular doxo about 1 h after addition in high extracellular amounts (as some sort of positive control). In case you may have problems seeing it, make sure to use a cancer cell line that responds with apoptosis to doxo. If you observe cell death after 24-32 h you at least know that doxo went in. Compare your signals to mock treated cells.

Importantly, time-lapse imaging is extremely messy with doxo, since it is a strong photosensitizer. For the fun of it, if using confocal, run a time-course over night. You'll most likely see that all cells in the field of view will be dead. When you move to a differetn region, cell death with be much lower or absent. As it seems that you are interested in early time points, this may not be a concern.

Have you considered using cell suspensions? Not sure how good it will work, but you may be able to pick up a signal spectroscopically.

Regarding the co-stains, there are supposedly specific nucleoli stains available that work in the basis of RNA binding. You can check the Invitrogen site, for example. I have never used them myself, though, and don't know whether what is available is compatible with your needs (spectrally). Some people use counterstaining of the nucleus as an option. E.g. Hoechst, and then stating that the dark areas in the nucleus are the nucleoli. I think that Hoechst 33258 and doxo may bind at differnt sites to the DNA, but you cannot exclude that they compete against each other, thereby distorting you measurements.

Endosomes can be visualised nicely by fusions of Rab proteins with GFP variants. All sorts of them have been published, and even cell lines stably expressing them may be available. Plenty of immunofluorescence options are available, if you want to work with fixed cells. Just check the literature.

thank you Markus , in case of live imaging and double stain ...is there any specefic medium to keep cells meanwhile ?

just regular growth medium, Rodi. Sometimes the phenol red in the medium may give you trouble with background signals, so that you may want to opt for phenol red-free medium (I usually don;t need it). Other than that, make sure you buffer your medium, e.g. with Hepes, or work with CO2 incubator on stage. A heated stage and objective is required for time lapse imaging, but for fixed time point reading you can ignore that. I understand that you are just going to take the cells out of the incubator to put them on stage for a single image.