RCC V was measured with a buffer containing 40 mM Tris; 10 mM ethylene glycol-bis(b-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), pH 8.0; 0.2 mM NADH; 2.5 mM phospho(enol)pyruvic acid monopotassium salt (Sigma-Aldrich); 2.5 [micro]g/mL antimycin A; 5 mM Mg[Cl.sub.2];5 U/mL lactate dehydrogenase (Sigma-Aldrich); 5 U/mL pyruvate kinase (Sigma-Aldrich); and 2.5 mM ATP (Sigma-Aldrich), which was supplemented with or without 2 [micro]g/mL oligomycin
(6 [micro]g/mL) was added to inhibit ATPase activity and verify F1F0-ATP synthase integrity; mitochondria were incubated with oligomycin
for 30 min .
, carbonyl cyanide-ptrifluoromethoxyphenylhydrazone (FCCP), and rotenone combined with antimycin A were all prepared to result in a 4 [micro]M final concentration after injection.
coli LPS (serotype 0127:B8), Leibovitz L15 medium, oligomycin
, FCCP (carbonyl cyanide-p- trifluoromethoxyphenylhydrazone), rotenone, antimycin, myxothiazol, a-tocopherol, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,7-dichlorofluoresceindiacetate (DCF-DA), and lipid standards were from SigmaAldrich (St.
complex was purchased from Cayman Chemical Co., quinidine and 4-aminopyridine were purchased from TCI.
, carbonyl cyanide-4(trifluoromethoxy) phenylhydrazone (FCCP), and antimycin A/rotenone were separately injected in each drug port in the sensor cartridge+utility plate and incubated in a C[O.sub.2]-free incubator for 10 min.
Different responses to treatment with oligomycin
and with the FAO inhibitor etomoxir were observed by both MRS and confocal microscopy and were related to GSC heterogeneity.
State 3 was initiated with ADP (0.5 mM), while state 4 was induced with the addition of oligomycin
Uncoupled respiration (State 4) was induced by the administration of oligomycin
During the subsequent course of measurements, four compounds were injected, in the following order: ADP (4mM), oligomycin
A (2.5 [micro]g/mL), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 8 [micro]M), and antimycin A (8 [micro]M)--all final concentrations.
After recording basalrespiration, oligomycin
(0.1 mg/mL) (Thermo Scientific, Waltham, MA, USA) was added to detect uncoupling respiration.
Thereafter, 50 [micro]L of the test reagents were sequentially injected into each well to obtain a final concentration of 4 [micro]M for each of the following: oligomycin
(Sigma-Aldrich, Germany), carbonyl cyanide 3-chlorophenylhydrazone (CCCP, Sigma), and rotenone/antimycin mixture (Sigma).