Dear colleagues,
I am trying to determine mitochondrial membrane potential with the help of TMRE but it's not working. I am following the protocol from abcam website which is incubating the cells with 500 nM TMRE in 96-well plate for 30 minutes and then measuring the fluorescence at (ex/em) 549/575. The plate-reader is not detecting any signal. The funny is that the fluorescence of the blank wells, i-e., 0.2% BSA in PBS, is higher than the wells with cells treated with TMRE. Please help me out here.
Thanks!
Are you using black-wall microplates, as recommended? Stray light could be a problem with clear plates. Did you replace the medium with PBS + 0.2% BSA? Were you careful not to allow the TMRE to become hydrolyzed before applying it to the cell culture?
Hi Adam!
Thanks for replying.
Yes, I used black-wall plates and replaced the staining medium with PBS having 0.2% BSA. I made a fresh stock of TMRE in DMSO just before adding to the cells. What precautions should I take to prevent hydrolysis of the dye? The DMSO I used has been sitting on the shelf for ages, do you think it has higher water content than it is supposed to be?
Thanks.
DMSO does absorb moisture, but if you made the solution of TMRE immediately before using it, it should have been OK.
I'm also using this protocol recently, but I didn't repeat aspirate PBS after the first adding. The signal was good but the FCCP was even higher than cells untreated. Do you have some ideas about improving it.
Thanks.
- Badges
- Science topic
- Similar topics
- Methodology
- Laboratory
- Cellular Biology Techniques