Good morning. I am working on cell cultures of neuroblastoma SHSY5Y and in order to evaluate the effect of a protein on the mitochondrial membrane, I thought about performing a functional test. Specifically I want to perform a cytofluorimetric measurement of the membrane potential. I used tmrm 150nM, for 30 minutes in incubation at 5%CO2. The probe gave no signals. I tried to use Mitotracher 350 nM under the same conditions but on cells in suspension and I got a fluorescence signal. The problem is that as a positive control I used a well-known decoupling, FCCP. I tried different concentrations from 1uM to 100 uM for both 1H of incubation and 24 H. However, I was unable to depolarize the membrane and achieve a decrease in fluorescence. Can you recommend a functional approach to perform the measurement?