Two mg of isolated mitochondria were suspended in a total volume of 1 ml consisting KCl buffer, 1 mM Malic acid (Sigma-Aldrich), 7 mM Pyruvate (Sigma-Aldrich), and 50 nM Calcium Green 5N (Invitrogen) in a quartz cuvette, which was placed inside the fluorimeter (PTI QuantaMaster 800, Horiba Scientific). Calcium uptake was measured by fluorescent emission of Calcium Green 5N. Simultaneously, mitochondrial swelling was measured by transmittance light. Each BH3 mimetic was used at concentrations: 0.5, 1, 10, 50, 100 nM, and 1 µM. Final BH3 mimetics concentrations used throughout the study were 200 nM ABT-199 (Selleckchem.com , S8048), 100 nM A-1331852 (abcam, ab218112), 100 nM ABT-737 (EMD Millipore Sigma, 197333), 10 nM S63845 (Selleckchem.com , S8383), 10 nM Obatoclax (Selleckchem, GX15-070). For some experiments ADP (300 uM) (Sigma-Aldrich, A2754) and/or CsA (2 uM) (Sigma-Aldrich, 30024) were used to desensitize the MPTP. CaCl2 (20 μM, 40 μM, and 80 µM) (Sigma-Adrich, C4901) was added into this system in succession until MPTP opening occurred, indicated by an upturn in Calcium Green 5N fluorescence, or when mitochondria were saturated with Ca2+ and were no longer able to take up further additions of CaCl2, indicated by a stair-stacking in the Ca2+ uptake graphs.
A2754
Manufactured by Merck Group
Sourced in United States
A2754 is a laboratory equipment product manufactured by Merck Group. It is a device designed for scientific research and analysis. The core function of A2754 is to perform specific tasks within a laboratory environment, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
C3755, by Merck Group (2 mentions)
V 650, by Jasco (2 mentions)
A2383, by Merck Group (2 mentions)
Malic acid, by Merck Group (1 mentions)
Calcium green 5n, by Thermo Fisher Scientific (1 mentions)
Pyruvate, by Merck Group (1 mentions)
Abt 199, by Selleck Chemicals (1 mentions)
Obatoclax, by Selleck Chemicals (1 mentions)
Pti quantamaster 800, by Horiba (1 mentions)
A 1331852, by Abcam (1 mentions)
Calcium green 5n, by Merck Group (1 mentions)
Kx 21n, by Sysmex (1 mentions)
Digitonin, by Merck Group (1 mentions)
Oxygraph, by Hansatech (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Phosphocreatine, by Merck Group (1 mentions)
P7936, by Merck Group (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Diva software 6, by BD (1 mentions)
D7008, by Merck Group (1 mentions)
12 protocols using a2754
1
Mitochondrial Calcium Uptake and Swelling Assay
2
Platelet Activation and Aggregation Assay
3
Mitochondrial Respiration in BMDMs
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0.5x105 BMDMs were seeded in a 96-well Seahorse plate. Following adherence, cells were washed and incubated in a mitochondrial assay solution (MAS) comprised of 220 mM mannitol, 70 mM sucrose, 5 mM MgCl2, 10 mM KH2PO4, 2 mM HEPES, 1 mM EGTA, and 0.2% (w/v) fatty acid free BSA at pH 7.4. Immediately prior to the start of the assay, cells were incubated with MAS supplemented with 4 mM ADP (A2754, Sigma) and 10 µg/ml digitonin (D141, Sigma). OCR measurements of digitonin-permeabilized cells were made before and after the addition of 1.25 mM succinic acid to cells and relative metabolic response to succinate was derived from these measures.
4
Measuring Mitochondrial Respiration in Spermatozoa
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The mitochondrial respiratory activity of spermatozoa was evaluated with a polarographic assay of oxygen consumption performed in sperm cells. Polarography measures the rate of change of oxygen concentration in a solution and, since oxygen is the last in the respiratory chain to accept electrons, provides a direct measure of mitochondrial activity [38 (link)].
Oxygen uptake by rat spermatozoa (2 × 107 sperm cells) was measured by using a Clark-type oxygen probe (Hansatech oxygraph; Hansatech Pentney, King’s Lynn, UK). Oxygen consumption was measured in the presence of a solution of respiratory substrates (10 mmol/L pyruvate and 10 mmol/L malate) and 0.76 μmol/L of adenosine diphosphate (ADP) (107360, 240176 and A2754, Sigma Aldrich).
Sperm mitochondria respiration efficiency was measured using the RCR, which was calculated by dividing rate of oxygen uptake measured in the presence of substrates + ADP (V3) by rate of oxygen uptake measured with substrates alone (V4) [10 (link)].
Oxygen uptake by rat spermatozoa (2 × 107 sperm cells) was measured by using a Clark-type oxygen probe (Hansatech oxygraph; Hansatech Pentney, King’s Lynn, UK). Oxygen consumption was measured in the presence of a solution of respiratory substrates (10 mmol/L pyruvate and 10 mmol/L malate) and 0.76 μmol/L of adenosine diphosphate (ADP) (107360, 240176 and A2754, Sigma Aldrich).
Sperm mitochondria respiration efficiency was measured using the RCR, which was calculated by dividing rate of oxygen uptake measured in the presence of substrates + ADP (V3) by rate of oxygen uptake measured with substrates alone (V4) [10 (link)].
5
Xenopus Egg Extract Replication Assay
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HSS and NPE were prepared as described previously (24 (link)) and stored at −80°C. Before both bulk- and single-molecule replication assays, each 33-μl aliquot of HSS was supplemented with 250 ng of nocodazole (Sigma-Aldrich, M1404) and 1 μl of an adenosine triphosphate (ATP) regeneration system, containing 650 mM phosphocreatine (Sigma-Aldrich, P7936), 65 mM ATP (pH 7.0; Sigma-Aldrich, A2754) and creatine phosphokinase (0.161 mg/ml; Sigma-Aldrich, C3755). Similarly, each 16-μl aliquot of NPE was supplemented with 0.5 μl of ATP mix. Activated extracts were centrifuged for 5 min at 16,000g, room temperature and used in replication assays. All Xenopus work fully complied with the UK Animals (Scientific Procedures) Act 1986 as implemented by the Francis Crick Institute.
6
Platelet Aggregation and ROS Measurement
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PRP samples (adjusted to 2 × 107 platelets/mL) were stimulated with the adenosine 5′-diphosphate (ADP, 16.7 µM, Sigma Aldrich A2754, Burlington, MA, USA) or phorbol 12-myristate 13-acetate (PMA, 100 µM, Sigma Aldrich 79346, Burlington, MA, USA). For ADP stimulation, the recording time was 10 min, and for PMA, the recording time was 15 min (AgreGO, Sao Paulo, Brazil). The degree of aggregation was expressed as a percentage of the maximum light transmission obtained with PRP [23 (link)].
DHE probe (500 µM, Sigma Aldrich D7008, Burlington, MA, USA) was added to 200 µL of the platelet suspension, protected from light, and placed at 37 °C for 30 min. After washout, the fluorescence was evaluated with a flow cytometer FACS CANTO II equipped with a 15-mW argon laser, λ = 488 nm (BD, Santa Monica, CA, USA). A total of 10,000 events were acquired in the FITC channel (564–606 nm) [21 (link),22 (link)], followed by analysis using the DIVA software 6.0 (BD, Santa Monica, CA, USA).
DHE probe (500 µM, Sigma Aldrich D7008, Burlington, MA, USA) was added to 200 µL of the platelet suspension, protected from light, and placed at 37 °C for 30 min. After washout, the fluorescence was evaluated with a flow cytometer FACS CANTO II equipped with a 15-mW argon laser, λ = 488 nm (BD, Santa Monica, CA, USA). A total of 10,000 events were acquired in the FITC channel (564–606 nm) [21 (link),22 (link)], followed by analysis using the DIVA software 6.0 (BD, Santa Monica, CA, USA).
7
Spectroscopic Determination of Nucleotide Stocks
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Both ADP and ATP stocks were prepared from their respective salt hydrates (nos. A2754 and A2383, Sigma Aldrich), supplemented with an equal molar amount of MgCl2 and adjusted to pH 7.5 with 1 M Tris-HCl. Final nucleotide concentration was spectroscopically determined (wavelength λ = 259 nm; V-650, Jasco) using an extinction coefficient of 15,400 M–1 cm−1.
8
Kras-Induced Zebrafish Larval Treatment
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Both krasV12 induction and chemical treatments were conducted in larvae from 3 dpf to 7 dpf. The chemicals used included doxycycline (dox) (20 μg/ml; D9891; Sigma), cilengitide (10 μm; SML1594; Sigma) and ADP (Adenosine 5′-diphosphate) (10 μm; A2754; Sigma). The dosages were selected based on the highest all-survival concentrations as determined in preliminary experiments [17] (link), [18] (link).
9
Seahorse Metabolic Profiling of H9c2 Cells
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Oxygen consumption was measured in H9c2 cells using a Seahorse XF Analyzer (Agilent Technology, Santa Clara, CA) as described before (Dott et al, 2014 (link)). H9c2 cells were seeded in Seahorse cell culture plates (Agilent Technology, Santa Clara, CA). Basal oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured to establish baseline rates. The cells were then treated with the following compounds in succession: 1 µg/mL oligomycin (Sigma, O4876), 0.5 µg/mL FCCP (Sigma, C2920), and 1 µg/mL rotenone (Sigma, R8875). Mitochondrial respirometry by Seahorse was conducted by an operator in a blinded fashion.
For the measurement of oxygen consumption in permeabilized cardiac fibers, left ventricular (LV) subendocardial muscle fibers were used to measure oxygen consumption rates and ATP synthesis as previously described (Boudina et al, 2005 (link)). Oxygen consumption rates were determined in the presence of palmitoyl-carnitine (Sigma, P1645) at 0.02 mM (VO), 1 mM ADP (A2754, Sigma) (VADP), and 1 μg/mL of the ATP synthase inhibitor oligomycin (A7699, Sigma) (VOligo). ATP levels were measured in fibers after the addition of 1 mM ADP.
For the measurement of oxygen consumption in permeabilized cardiac fibers, left ventricular (LV) subendocardial muscle fibers were used to measure oxygen consumption rates and ATP synthesis as previously described (Boudina et al, 2005 (link)). Oxygen consumption rates were determined in the presence of palmitoyl-carnitine (Sigma, P1645) at 0.02 mM (VO), 1 mM ADP (A2754, Sigma) (VADP), and 1 μg/mL of the ATP synthase inhibitor oligomycin (A7699, Sigma) (VOligo). ATP levels were measured in fibers after the addition of 1 mM ADP.
10
Enzymatic Assays for Creatine and Creatine Phosphate
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The assays for creatine and creatine phosphate were based on the enzymatic reactions:
Creatine and creatine phosphate were measured by the consumption of NADPH measured by absorption at 340 nm at 37°C. Extracted sample was suspended in a working solution (200 mM phosphate buffer [200 mM KH2PO4 (P5504; Sigma) and 200 mM Na2HPO4 (4062-01; JT Baker, Phillipsburg, NJ)], 7 mM MgCl2 (M0250; Sigma), 1.8 mg NADPH (Calzyme), 10 mM of α-ketoglutarate (75892; Sigma), and 2.5 mM ADP (A2754; Sigma) and the reactions were catalyzed by adding 6 U of glutamate dehydrogenase (GLDH; G4387; Sigma), 100 U of urease (U1875; Sigma), and 90 U of creatinase (C2409; Sigma). After creatine is consumed, creatine phosphate was measured by adding 42 U creatine kinase (CK; C3755; Sigma).
Creatine and creatine phosphate were measured by the consumption of NADPH measured by absorption at 340 nm at 37°C. Extracted sample was suspended in a working solution (200 mM phosphate buffer [200 mM KH2PO4 (P5504; Sigma) and 200 mM Na2HPO4 (4062-01; JT Baker, Phillipsburg, NJ)], 7 mM MgCl2 (M0250; Sigma), 1.8 mg NADPH (Calzyme), 10 mM of α-ketoglutarate (75892; Sigma), and 2.5 mM ADP (A2754; Sigma) and the reactions were catalyzed by adding 6 U of glutamate dehydrogenase (GLDH; G4387; Sigma), 100 U of urease (U1875; Sigma), and 90 U of creatinase (C2409; Sigma). After creatine is consumed, creatine phosphate was measured by adding 42 U creatine kinase (CK; C3755; Sigma).
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