atp synthesis buffer  (Thermo Fisher)

Journal: Scientific Reports

Article Title: Identification of ER-000444793, a Cyclophilin D-independent inhibitor of mitochondrial permeability transition, using a high-throughput screen in cryopreserved mitochondria

doi: 10.1038/srep37798

Figure Lengend Snippet: ( a ) Representative oxygen flux recording using closed-chamber, high-resolution respirometry. Rat liver mitochondria (0.25 mg protein ml −1 ) were maintained under constant stirring. Oxygen consumption (black trace) and oxygen concentration (grey trace) were measured using the Oxygraph 2 K (Oroboros Instruments, Innsbruck, Austria). Sequential additions of reagents were added to assess respiratory states. ( b ) Mitochondria energised using either glutamate/malate (10 mM/2 mM; black trace) or succinate and rotenone (10 mM/1 μM; grey trace) were treated with oligomycin A (2.5 μg ml −1 ) and FCCP (0.5 μM titration) to assess leak respiration and spare respiratory capacity respectively. ( c ) RCR (respiratory control ratio, calculated as the ratio of State 3/State 4 respiration) for freshly isolated mitochondria and trehalose freeze-thawed mitochondria from the same preparation energised using glutamate/malate (10 mM/2 mM). Statistical significance was calculated using a one-way ANOVA corrected for multiple comparisons using Tukey method (NS P > 0.05; GraphPad Prism). ( d ) RCR for 3 month cryopreserved mitochondria energised using glutamate/malate (10 mM/2 mM). ( e ) Mitochondria (1 mg protein ml −1 ) were incubated in the presence of 1 mM ADP and either glutamate/malate (10 mM/2 mM) or succinate/rotenone (10 mM/1 μM) and total ATP content was measured after 45 minutes. Luminescence was measured in the presence and absence of respiratory inhibitors antimycin A (2.5 μM) and oligomycin A (2.5 μg ml −1 ) to determine background. Data are expressed as means (±s.d.) of at least three independent experiments using mitochondria that had been cryopreserved for between 1 and 3 months. Abbreviations: Mito; mitochondria, Glu; glutamate, Mal; malate, Succ; succinate, FCCP; carbonyl cyanide-p-trifluoromethoxyphenylhydrazone.

Article Snippet: Mitochondria (1 mg protein ml −1 final concentration) were re-suspended in ATP synthesis buffer (20 mM Tris base 0.6 M sorbitol, 15 mM potassium phosphate monobasic, 10 mM magnesium sulphate, 2.5 mg ml −1 essentially fatty acid free BSA, final pH 7.4) containing either 10 mM L-glutamic acid, monosodium salt; 2 mM L-malic acid sodium salt or 10 mM succinate disodium salt; 1 μM rotenone and mitochondrial suspension (20 μl) was dispensed into a clear bottom black-walled 384 well plate using a Multidrop Combi Reagent Dispenser (Thermo Scientific, Rockford, IL).

Techniques: Concentration Assay, Titration, Control, Isolation, Incubation

Journal: Scientific Reports

Article Title: Identification of ER-000444793, a Cyclophilin D-independent inhibitor of mitochondrial permeability transition, using a high-throughput screen in cryopreserved mitochondria

doi: 10.1038/srep37798

Figure Lengend Snippet: ( a,b ) Rat liver mitochondria (1 mg protein ml −1 ) were incubated with compound for 10 minutes in the presence of either glutamate (10 mM) and malate (2 mM) or succinate (10 mM) and rotenone (1 μM). ADP (5 mM) was added and incubated for 45 minutes. Mitochondria were lysed and total ATP quantified by luminescence using CellTiter Glo reagent (Promega, Madison, WI). No effect of either ER-000444793, CsA or SfA was observed under either respiratory substrate condition. Results are expressed as % inhibition of ATP, synthesis normalised to DMSO (0% inhibition) and antimycin A (2.5 μM) plus oligomycin A (2.5 μg ml −1 ; 100% inhibition). Data are expressed as means (± s.d.) of at least three independent experiments. ( c ) Assessment of immunosuppression and cellular toxicity in Jurkat NFAT-reporter cells. Jurkat cells were incubated with compound for 45 minutes prior to the addition of ionomycin (0.5 μM) and PMA (50 nM). NFAT reporter activity was assessed after 20 hours treatment with either ER-000444793, CsA or SfA and quantified by luminescence using Bright Glo reagent (Promega, Madison, WI). Data are expressed as % inhibition, normalised to DMSO (0% inhibition) and CsA (5 μM; 100% inhibition). ( d ) Toxicity of ionomycin/PMA treatment was assessed in Jurkat cells using Alamar blue after 20 hours incubation. No significant effect was observed with treatment. Data are expressed as fluorescence intensity and statistical significance calculated using paired two-tailed student’s t -test using GraphPad Prism (NS P > 0.05). ( e ) Jurkat cells were incubated with compound for 20 hours and toxicity assessed using Alamar Blue. Data are expressed as fluorescence intensity and compared using multiple t-tests comparing treatments to DMSO, corrected for multiple comparisons using Holm-Sidak method , P < 0.05 defined as being statistically significant. All data are expressed as means (± s.d.) of three independent experiments. Abbreviations: CsA; cyclosporin A, SfA; sanglifehrin A, Cmp; compound, O.D; optical density, NS; not significant, PMA; phorbol 12-myristate.

Article Snippet: Mitochondria (1 mg protein ml −1 final concentration) were re-suspended in ATP synthesis buffer (20 mM Tris base 0.6 M sorbitol, 15 mM potassium phosphate monobasic, 10 mM magnesium sulphate, 2.5 mg ml −1 essentially fatty acid free BSA, final pH 7.4) containing either 10 mM L-glutamic acid, monosodium salt; 2 mM L-malic acid sodium salt or 10 mM succinate disodium salt; 1 μM rotenone and mitochondrial suspension (20 μl) was dispensed into a clear bottom black-walled 384 well plate using a Multidrop Combi Reagent Dispenser (Thermo Scientific, Rockford, IL).

Techniques: Incubation, Inhibition, Activity Assay, Fluorescence, Two Tailed Test