Talk:Digitonin
Anna Pakula
Dear MiPNet-members,we work with C2C12 cells and 3T3 cells on Oxygraph Oroboros 2k. The amount of cells is: 1 million cells in 2ml respiration buffer: MIRO5 and/or medium D (Hepes 200mM, KH2PO4 125mM, MgCl2 100mM, sucrose 2,5M according to protocol Yan Lab by Ying (Lynn) Li, 07.12.2013). Cells are trypsinized with 0,05% trypsin-EDTA and centrifuged (800rpm, 3 min). After counting the cells and trypan blue test ( ~95% cells were alive), we added into the pellet respiration buffer with 3ul digitonin ( for 1 mln cells, stock 10mg/ml) and put it into chambers. We increased gradually digitonin concentrations to 9ul or even more, without seeing any differences. The O2 concentration max reached ~180 [mmol/ml] at the start of experiment. The problem (for both C2C12 and 3T3 cells) we have is following: after addition of substrates (concentrations and order according to Kuznetsov A et al, 2008 protocol) we did not see any kind of changes in the respiration rate at all. After taking out the cells from chambers their permeabilization was checked by Trypan blue staining and cells were positive stained after the experiment. After many tests we think there is a problem with digitonin and think to replace it with saponin. If somebody used to work with both of them what concentration do you think will be worth to start experiment with? We did not wash cells to remove digitonin after the permeabilization as it was suggested by Fischer-Wellman KH, et al. 2013) and we are not sure if it could have such a big meaning for our cells? What do you think might be the major problem that our experiments do not work at all? We will be thankful for any suggestions from your side. Thank you in advance
Alexandra Latini
Your chambers are not contaminated with inhibitors, right? It happened to me many times, even when they were exhaustively cleaned up.
Bernd Schoepf
I am using Digitonin to permeabilize my PCa & BCa cell lines (PC3, LNCaP, RWPE1 and some more), but I never had any problems so far.... Could you maybe provide or post a trace so that we have more details? And if you maybe post the exact SUIT and washing procedures after your experiments we could also elaborate on that (e.g. saturating concentrations, inhibitor carry overs etc). What concentration/ammount did you use (microgramms per 10^6 cells)? Which stock solution did you use? Note that it will take some time for Digitonin to fully permeabilize the membrane! You can see it nicely when starting from (15 min of) Routine state (R). You should reach Leak state (L) within some other 10 to 15 minutes. Did you perform a titration experiment (adding Dig stepwise after substrates + ADP)? If not, I can mail you one of my traces and the SUIT. I suggest you run your rxperiments under normoxic conditions?
