I have tried three different concentrations for doxorubicin, 10uM, 50 uM, 100 uM for 2 hrs. The effect attained was similar at all three concentrations, but there was a problem while conducting an image acquisition. All the analogs were stained well with hoechst at 1ug/ml except in doxorubicin treated wells. Somehow its masking the intensity level of hoechst even at 5 ug/ml.
Should I lower the doxorubicin concentration or is there any other alternative?
Are you looking at growth inhibition with doxorubicin or radical formation or ???? In spite of the concept of reproducibility, cells behave differently in different labs with different users and different passages. Hoechst will stain the nuclei but doxorubicin will also find its way to the nucleus and depending on whatever wavelength you're looking at, you're probably getting some disturbance from doxorubicin in the nucleus. You could look at the wavelengths for Hoechst and doxorubicin but you may have to alter your experimental readout or change your interpretation to confirming the presence of doxorubicin in the nucleus if you see this interference with Hoechst....
which cells you are incubating? and what is the aim of different concentrations? do you want to reach IC50?
Doxorubicin has an IC50 of about 100 nM. Hence, you should use between 500 nM to 1 micromolar to get best effect. At higher concentrations prozone effect sets in and you may not get the desired effect.
Moustapha Hassan...we are using THP1 cells . Doxorubicin is set up as a positive control to produce maximum ROS production.
Vidhya Iyer ...i am using Doxo as a positive control for maximum ROS production. rest everything is fine except that as the free drug disperses around the nucleus as well and it having the same excitation range as that of hoechst is overlapping with the blue stain. Therefore while analysing the images via automated fashion , doxorubicin treated cells are left uncounted
Please check: Wang et al. 2004. J. Biol. Chem. vol 279, pg 25535. They used 100 nM to 500 nM Doxorubicin to induce oxidative stress. My recommendation is still to reduce the amount of DOX you are using. Give it a try.
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