Hello everybody,

I have a question about ATP depletion. I have seeded my tumor cell line on glas coverslips and treated them with oligomycin B (50µM, 4h). Before I have started my experiment I removed the medium with glucose and replaced it with glucose-free medium. After the incubation time and before fixation I wanted to wash my cells mit PBS. Unfortunately I washed all cells away. I could fix just a few cells. Okay, then I reduced the concentration 5µM for an hour. The same result.
Who knows a concentration of oligomycin b for MCF-7? It is very urgent.
I am grateful for all comments.
Thanks in advance.
Tiffy

Hi Tiffy! As far as I know, oligomycin is used in the low uM or even sub-uM range (0.1-5 uM) for 3-4h. Where did you get the 50 uM protocol from?
Also, try using PBS+Ca+Mg+0.5% albumin (BSA) for your washes - do those very gently: I found that washing coverslips in a 12-well plate, adding and aspirating PBS down the side-wall, really helps.

Hey samm,

I found the conc. at Reilley et al JCB 2006. Well, they used 3T3 but I can`t imagine that these cells were more robust. Luckily I found a publication where the authors show that you can avoid the lost of cell adhesion with HGF. I will try it.
Do you use deoxyglucose as well?
Regards
Tiffy

Actually, I've observed that 3T3s tend to come away even more easily - the great thing about them is their transfection potential! I have never used HGF or deoxyglucose. However, I would strongly recommend that you try titering out oligomycin from ~0.02 to 2.5 uM in 5-fold intervals (0.02; 0.1; 0.5; 2.5 uM - the 3 h treatment is pretty standard) to find an optimal concentratio for your batch of MCF7s, as well as use the gentle wash with D-PBS containing Ca and Mg.
All the best
-sam