I am having problems with my protocol. I am trying to optimize a protocol to assess mitochondrial function in gastric mucosa using the Oroboros. Any tips?
Hi Cassilda
My work on cardiac mitochondria and permeabilized fibers was extensive , but no experience of working on gut mucosa. If you mail me your protocol, I am willing t try and trouble shoot for you ? Dr J McCarthy, [email protected]
Thank you for your interest Dr J McCarthy.
So my protocol is:
-Biopsies from the gastric mucosa of rats is recovered and paleced in ice cold solution (in mM) CaK2EGTA 2.77, K2EGTA 7.23, MgCl2 6.56, DTT 0.5, K-MES 50, imidazole 20, taurine 20, Na2ATP 5.3, phosphocreatine 15, at pH 7.1.
-Permeabilization with 50ug/ml saponin for 30 min 4ºC.
-washed in respiration medium Mir05, 110 mM sucrose,
50 mM K -MES, 0.5 mM EGTA, 3 mM MgCl 2 , 20 mM
taurine, 10 mM KH 2 PO 4 , 20 mM HEPES adjusted to pH 7.1
with KOH at 37 °C; and 1 g/l BSA essentially fatty acid free for 10 min , 3x.
-oxigen consumption assed by Oroboros in Mir05 respiration medium 37ºC
It is very difficult to get a response to substrates such as glutamate/malate ou even glucosa and pyruvate,with or without ADP, only succinate works (some times), Oligomycin does not inhibits respiration and FCCP can not stimulate oxigen comsumption. I tried a lot of concentrations and brands!
Do you some hint for me?
Thank you for your attention!
the method I used is from a ref in Circulation: 2012 by Sahim Boudina ... Mito abnormalities in Obsity. the resp buffer is similar , but I tested permemabilzation times by exposing the cardiac fibers to 2 min , 3 , 5 10 a20 and 20mins and found that 20 mins on ice with 50mg/ml saponon worked optimally - shorter or longer was not as effective, Perhaps you do not need 30 mins ?? Sounds long for a relatively delicate tissue. Remember that the saponin is non-specific and once it's through the cell membrane ,it will permeabilize every membrane it encounters thereafter , incl the mito ...
So try a time trial, as I did and see if shorter exposure to saponin improves yr respiration.
Thank you for your input!
I will try a shorter exposure to saponin.
Hi Cassilda,
To check if the mitochondrial membrane has been altered by the saponin exposition you can use a cytochrome c tritation. If the active respiration (Routine respiration with oroboros) rises more than a 10% is indicative of an altered mtiochondria. I do not know if that could help to set up your incubation period with the saponin.
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